Biaryl compositions in combination with tamoxifen and methods for modulating a kinase cascade

ABSTRACT

The invention relates to compounds and methods for modulating one or more components of a kinase cascade.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/232,175, filed on Dec. 26, 2018, which is a division of U.S.application Ser. No. 12/595,357, filed on Apr. 30, 2010 (now U.S. Pat.No. 10,196,357), which is a national stage application, filed under 35U.S.C. § 371, of International Application No. PCT/US2008/004847, filedon Apr. 14, 2008, which claims the benefit of U.S. Patent ApplicationSer. No. 60/923,496, filed on Apr. 13, 2007. The entire contents of theabove-identified applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Signal transduction is any process by which a cell converts one kind ofsignal or stimulus into another. Processes referred to as signaltransduction often involve a sequence of biochemical reactions insidethe cell, which are carried out by enzymes and linked through secondmessengers. In many transduction processes, an increasing number ofenzymes and other molecules become engaged in the events that proceedfrom the initial stimulus. In such cases the chain of steps is referredto as a “signaling cascade” or a “second messenger pathway” and oftenresults in a small stimulus eliciting a large response. One class ofmolecules involved in signal transduction is the kinase family ofenzymes. The largest group of kinases are protein kinases, which act onand modify the activity of specific proteins. These are used extensivelyto transmit signals and control complex processes in cells.

Protein kinases are a large class of enzymes which catalyze the transferof the γ-phosphate from ATP to the hydroxyl group on the side chain ofSer/Thr or Tyr in proteins and peptides and are intimately involved inthe control of various important cell functions, perhaps most notably:signal transduction, differentiation, and proliferation. There areestimated to be about 2,000 distinct protein kinases in the human body,and although each of these phosphorylate particular protein/peptidesubstrates, they all bind the same second substrate, ATP, in a highlyconserved pocket. Protein phosphatases catalyze the transfer ofphosphate in the opposite direction.

A tyrosine kinase is an enzyme that can transfer a phosphate group fromATP to a tyrosine residue in a protein. Phosphorylation of proteins bykinases is an important mechanism in signal transduction for regulationof enzyme activity. The tyrosine kinases are divided into two groups;those that are cytoplasmic proteins and the transmembranereceptor-linked kinases. In humans, there are 32 cytoplasmic proteintyrosine kinases and 58 receptor-linked protein-tyrosine kinases. Thehormones and growth factors that act on cell surface tyrosinekinase-linked receptors are generally growth-promoting and function tostimulate cell division (e.g., insulin, insulin-like growth factor 1,epidermal growth factor).

Inhibitors of various known protein kinases or protein phosphatases havea variety of therapeutic applications. One promising potentialtherapeutic use for protein kinase or protein phosphatase inhibitors isas anti-cancer agents. About 50% of the known oncogene products areprotein tyrosine kinases (PTKs) and their kinase activity has been shownto lead to cell transformation.

The PTKs can be classified into two categories, the membrane receptorPTKs (e.g. growth factor receptor PTKs) and the non-receptor PTKs (e.g.the Src family of protooncogene products). There are at least 9 membersof the Src family of non-receptor PTK's with pp60^(c-src) (hereafterreferred to simply as “Src”) being the prototype PTK of the familywherein the approximately 300 amino acid catalytic domains are highlyconserved. The hyperactivation of Src has been reported in a number ofhuman cancers, including those of the colon, breast, lung, bladder, andskin, as well as in gastric cancer, hairy cell leukemia, andneuroblastoma. Overstimulated cell proliferation signals fromtransmembrane receptors (e.g. EGFR and p185HER2/Neu) to the cellinterior also appear to pass through Src. Consequently, it has recentlybeen proposed that Src is a universal target for cancer therapy, becausehyperactivation (without mutation) is involved in tumor initiation,progression, and metastasis for many important human tumor types.

Because kinases are involved in the regulation of a wide variety ofnormal cellular signal transduction pathways (e.g., cell growth,differentiation, survival, adhesion, migration, etc.), kinases arethought to play a role in a variety of diseases and disorders. Thus,modulation of kinase signaling cascades may be an important way to treator prevent such diseases and disorders.

SUMMARY OF THE INVENTION

Compounds of the invention are useful in modulation a component of thekinase signaling cascade. Some compounds may be useful in modulation ofmore than one component of a kinase signaling cascade. The compounds ofthe present invention are useful as pharmaceutical agents. The compoundsof the invention may be useful for modulating regulation of a kinasewhich may be involved in a normal cellular signal transduction pathway(e.g., cell growth, differentiation, survival, adhesion, migration,etc.), or a kinase involved in a disease or disorder. Such diseasesinclude, without limitation, cancer for example renal, liver, and braincancer.

The compounds of the invention are useful in treating diseases anddisorders that are modulated by tyrosine kinase inhibition. For example,the compounds of the invention are useful in treating diseases anddisorders that are modulated by Src kinase. The compounds of theinvention may also be useful in treating diseases and disorders that aremodulated by focal adhesion kinase (FAK).

The invention provides for a method of treating or preventing cancer,comprising administering a compound of the invention to a subject,wherein the cancer is selected from renal, liver, and brain cancer. Theinvention provides for use of a compound according to Formula IB:

or a salt, solvate, hydrate, or prodrug thereof, wherein:

T is a bond;

X_(y) is CY, N, or N—O;

X_(z) is CZ;

Y is selected from hydrogen, hydroxyl, halogen, lower (C₁, C₂, C₃, C₄,C₅, or C₆) alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃,C₄, C₅, or C₆) alkyl-aryl, and O-benzyl;

X_(a) is CR_(a) or N, or N—O;

X_(b) is CR_(b), N, or N—O;

X_(c) is CR_(c) or N, or N—O;

X_(d) is CR_(d) or N, or N—O;

X_(e) is CR_(e), N, or N—O;

R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ are, independently,hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂,C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆)alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂-lower (C₁, C₂,C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl;

P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower alkyl is linear orbranched alkyl;

K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or;

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are C₁, C₂, C₃, C₄, C₅, or C₆ alkyl or R₁₉ and R₂₀taken together with the attached nitrogen atom form a five memberedring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—;

R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently, H or C₁, C₂,C₃, C₄, C₅, or C₆ alkyl;

Z is (CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, R₁, R₂, andR₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; and

n and m are independently 0, 1, or 2,

in the manufacture of a medicament for treating or preventing cancer,wherein the cancer is selected from renal, liver, or brain cancer.

In one embodiment, the invention provides the use, wherein themedicament comprises at least one pharmaceutically acceptable excipientor carrier.

In one embodiment, the invention provides the use, wherein the cancer isbrain cancer. In another embodiment, the invention provides the use,wherein the brain cancer is a primary tumor. In another embodiment, theinvention provides the use, wherein the primary brain tumor is selectedfrom glioblastoma, astrocytoma, meningioma, pituitary adenoma,vestibular schwannoma, ependymona, oligodendroglioma, ependymomas,choroid plexus papilloma, and medullablastoma. In one embodiment, theinvention provides the use, wherein the cancer is neuroblastoma. In oneembodiment, the invention provides the use, wherein the cancer isneuroepitelioma.

In one embodiment, the invention provides the use, wherein the cancer isrenal cancer. In another embodiment, the invention provides the use,wherein the renal cancer is selected from renal cell carcinoma, renalpelvis carcinoma, Wilms tumors, clear cell carcinoma, renaladenocarcinoma, and renal rhabdomyosarcoma.

In another embodiment, the invention provides the use, wherein thecancer is liver cancer. In another embodiment, the invention providesthe use, wherein the liver cancer is selected from hepatocellularcarcinoma, cholangiocarcinoma, angiosarcoma, hemangiosarcoma, andhepatoblastoma.

In one embodiment, the invention provides the use, wherein at least oneof X_(a), X_(b), X_(c), X_(d), X_(e), X_(y) and X_(z) is N.

In another embodiment, the invention provides the use, wherein X_(z) isCZ and Z is

where R₇, R₈, R₉, R₁₀, and R₁₁ are selected from hydrogen, hydroxyl,halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁,C₂, C₃, C₄, C₅, or C₆ alkyl-OH, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-O—C₁,C₂, C₃, C₄, C₅, or C₆ alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl.

In another embodiment, the invention provides the use, wherein R₄ and R₆are each H.

In another embodiment, the invention provides the use, wherein at leastone of R₇, R₈, R₉, R₁₀ and R₁₁ is halogen, C₁, C₂, C₃, C₄, C₅, or C₆alkyl, or O-benzyl.

In another embodiment, the invention provides the use, wherein m and nare each 1 and R₂ and R₃ are each H.

In another embodiment, the invention provides the use, wherein thecompound is selected from

In another embodiment, the invention provides the use, wherein thecompound is a pharmaceutically acceptable salt.

In another embodiment, the invention provides the use, wherein thecompound is a hydrochloride salt.

The invention provides a composition comprising tamoxifen and a compoundof the invention. In one embodiment, the invention provides acomposition comprising tamoxifen and a compound of Formula I:

or a salt, solvate, hydrate, or prodrug thereof, wherein:

T is a bond, CR₁₂R₁₃, C(O), O, S, S(O), S(O)₂, NR₁₄, C(R₁₅R₁₆)C(R₁₇R₁₈),CH₂O, or OCH₂;

X_(y) is CZ, CY, N, or N—O;

X_(z) is CZ, CY, N, or N—O;

at least one of X_(y) and X_(z) is CZ;

Y is selected from hydrogen, hydroxyl, halogen, lower (C₁, C₂, C₃, C₄,C₅, or C₆) alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃,C₄, C₅, or C₆) alkyl-aryl, and O-benzyl;

X_(a) is CR_(a) or N, or N—O;

X_(b) is CR_(b), N, or N—O;

X_(c) is CR_(c) or N, or N—O;

X_(d) is CR_(d) or N, or N—O;

X_(e) is CR_(e), N, or N—O;

R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ are, independently,hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂,C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆)alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂-lower (C₁, C₂,C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl;

P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower alkyl is linear orbranched alkyl;

K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are C₁, C₂, C₃, C₄, C₅, or C₆ alkyl or R₁₉ and R₂₀taken together with the attached nitrogen atom form a five memberedring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—;

R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently, H or C₁, C₂,C₃, C₄, C₅, or C₆ alkyl;

Z is (CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, R₁, R₂, andR₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; and

n and m are independently 0, 1, or 2.

In one embodiment, the invention provides a composition comprisingtamoxifen and the compound

In another embodiment, the invention provides for use of the compositionof the invention in the manufacture of a medicament for treating orpreventing breast cancer. The invention provides a method of treating orpreventing breast cancer by administering to a subject tamoxifen and acompound of the invention. In one embodiment, the composition comprisesa compound of formula IB. In another embodiment, the ratio of thecompound and tamoxifen administered to a subject is not equal i.e., theratio is not 50:50. In one embodiment, the amount of tamoxifen in thecombination is greater than the amount of the compound of the invention.In another embodiment, the amount of the compound of the invention inthe combination is greater than the amount of tamoxifen.

Further, compounds may be used without limitation, for example, asanti-cancer, anti-angiogenesis, anti-metastatic, anti-microbial,anti-bacterial, anti-fungal, anti-parasitic and/or anti-viral agents.The compounds of the invention are useful, for example, in treating lungcancer. The compounds of the invention are also useful, for example, intreating colon cancer. The compounds of the invention are also useful,for example, in treating breast cancer.

Compounds of the invention include compounds of Formula I, and salts,solvates, hydrates, or prodrugs thereof:

where:

T is absent (i.e., the rings are connected by a bond), CR₁₂R₁₃, C(O), O,S, S(O), S(O)₂, NR₁₄, C(R₁₅R₁₆)C(R₁₇R₁₈), CH₂O, or OCH₂;

X_(y) is CZ, CY, N, or N—O;

X_(z) is CZ, CY, N, or N—O;

at least one of X_(y) and X_(z) is CZ;

Y is selected from hydrogen, hydroxyl, halogen, lower (C₁, C₂, C₃, C₄,C₅, or C₆) alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃,C₄, C₅, or C₆) alkyl-aryl, and O-benzyl;

X_(a) is CR_(a), N, or N—O;

X_(b) is CR_(b), N, or N—O;

X_(c) is CR_(c), N, or N—O;

X_(d) is CR_(d), N, or N—O;

X_(e) is CR_(e), N, or N—O;

R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ are, independently,hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂,C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆)alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl SO₂H, SO₂-lower (C₁, C₂, C₃,C₄, C₅, or C₆) alkyl, or

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl;P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl is linear or branched alkyl;K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, aryl,heteroaryl, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are independently C₁, C₂, C₃, C₄, C₅, or C₆ alkyl orR₁₉ and R₂₀ taken together with the attached nitrogen atom form a fivemembered ring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—;

R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently, H or C₁, C₂,C₃, C₄, C₅, or C₆ alkyl;

Z is (CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, such asbenzene, pyridine, or pyrimidine. For example, Z is;

where

R₁, R₂, and R₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl;

n and m are, independently 0, 1, or 2;

R₇, R₈, R₉, R₁₀, and R₁₁ are, independently, hydrogen, hydroxyl,halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁,C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH, COO-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl, SO₂H, SO₂-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl;

P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl is linear or branched alkyl;

K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, aryl,heteroaryl, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are independently C₁, C₂, C₃, C₄, C₅, or C₆ alkyl orR₁₉ and R₂₀ taken together with the attached nitrogen atom form a fivemembered ring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂—, or—OCH₂CH₂CH₂—.

In certain compounds of the invention, Z is

Certain compounds of the invention are selected from Compounds 1-136 and137. For example, the compound of the invention is Compound 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 93, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137.

Compounds of the invention include Compounds 33, 38, 40, 76, 133, 134,136 and 137.

Certain compounds of the invention are selected from Compounds 138-246and 247. For example, the compound of the invention is Compound 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, or 247.

Compounds of the invention include Compounds 146 and 147.

Certain compounds of the invention are selected from Compounds 248-273and 274. For example, the compound of the invention is Compound 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, or 274.

In certain Compounds of Formula I, at least one of X_(a), X_(b), X_(c),X_(d) and X_(e) is N.

For example, in the compound of Formula I, X_(a) is N and X_(b) isCR_(b), X_(c) is CR_(c), X_(d) is CR_(d) and X_(e) is CR_(e).

In certain compounds of Formula I, X_(y) is CY, and X_(z) is CZ.

For example, in certain compounds of Formula I, Y is hydrogen.

In certain compounds of Formula I, R_(b) is C₁, C₂, C₃, C₄, C₅, or C₆alkoxy. For example, R_(b) is methoxy or ethoxy. In certain compounds ofFormula I, R_(b) is hydrogen. In other compounds of Formula I, R_(b) isselected from F, Cl, Br, and I.

In other compounds of Formula I, R_(b) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. For example, V is a bond. In certaincompounds of Formula I, W is hydrogen. In other compounds of Formula I,W is C₁, C₂, C₃, C₄, C₅, or C₆ alkyl.

In certain compounds of Formula I, R_(c) is C₁, C₂, C₃, C₄, C₅, or C₆alkoxy. For example, R_(c) is methoxy or ethoxy. In other compounds ofFormula I, R_(c) is hydrogen, F, Cl, Br, or I.

In other compounds of Formula I, R_(c)

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—.

For example, V is a bond. In certain compounds of Formula I, W ishydrogen. In other compounds of Formula I, W is C₁, C₂, C₃, C₄, C₅, orC₆ alkyl.

In certain compounds of Formula I, R_(d) is C₁, C₂, C₃, C₄, C₅, or C₆alkoxy. For example, R_(d) is methoxy or ethoxy. In other compounds ofFormula I, R_(d) is hydrogen, F, Cl, Br, or I.

In other compounds of Formula I, R_(d) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—.

For example, V is a bond. In certain compounds of Formula I, W ishydrogen. In other compounds of Formula I, W is C₁, C₂, C₃, C₄, C₅, orC₆ alkyl.

The invention includes a solvate of a compound according to Formula I.

The invention also includes a hydrate of a compound according to FormulaI.

The invention also includes an acid addition salt of a compoundaccording to Formula I. For example, a hydrochloride salt e.g.,dihydrochloride.

The invention also includes a prodrug of a compound according to FormulaI.

The invention also includes a pharmaceutically acceptable salt of acompound of Formula I.

The invention also includes a composition of a compound according toFormula I and at least one pharmaceutically acceptable excipient.

The invention relates to a compound of Formula I, having a structureaccording to one of Formulae II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII.

or a salt, solvate, hydrate, or prodrug thereof, where: R_(b), R₄, R₅,R₈, and R₁₀ are, independently, hydrogen, hydroxyl, halogen, P, C₁, C₂,C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁,C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆alkyl-OH, COOH, COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H,SO₂-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl;P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl is linear or branched alkyl;K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, aryl,heteroaryl, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are independently C₁, C₂, C₃, C₄, C₅, or C₆ alkyl orR₁₉ and R₂₀ taken together with the attached nitrogen atom form a fivemembered ring; and

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—.

For example, in the compound of Formula II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII, R₈ is hydrogen, F, Cl, Br, or I. For example,R₈ is F. In certain compounds, R₈ is H.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R_(b) is C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy. Forexample, R_(b) is methoxy or ethoxy.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R_(b) is hydrogen, Cl, Br, or I. In other compounds,in the compound of Formula II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII, R_(b) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl, and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R₄ is hydrogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, F,Cl, Br, or I. In other compounds, in the compound of Formula II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII, R₄ is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R₅ is hydrogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, F,Cl, Br, or I. In other compounds, in the compound of Formula II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII, R₅ is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R₁₀ is hydrogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, F,Cl, Br, or I. For example, R₁₀ is methoxy, ethoxy or isobutoxy.

In other compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII, R₁₀ is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—.

For example, in the compound of Formula II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII, W is hydrogen, or C₁, C₂, C₃, C₄, C₅, or C₆alkyl.

Certain compounds of the invention include compounds according toFormula II.

The invention relates to a solvate of a compound according to one ofFormulae II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII. Theinvention also relates to a hydrate of a compound according to one ofFormulae II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII.

The invention also relates to an acid addition salt of a compoundaccording to one of Formulae II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. For example, a hydrochloride salt.

Further, the invention relates to a prodrug of a compound according toone of Formulae II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII.

The invention also relates to a pharmaceutically acceptable salt of acompound of one of Formulae II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIIII.

The invention includes compositions comprising a compound according toone of Formulae I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, andXIII and at least one pharmaceutically acceptable excipient.

The invention relates to compounds and methods of using the compounds tomodulate a component of the kinase signaling cascade. Some compounds maybe useful in modulation of more than one component of a kinase signalingcascade. The compounds of the present invention are useful aspharmaceutical agents.

Certain compounds of the invention are non-ATP competitive kinaseinhibitors.

The invention relates to compounds and methods of using the compounds totreat cell proliferation disorders.

The invention also includes a method of preventing or treating a cellproliferation disorder by administering a pharmaceutical compositionthat includes a compound according to one of Formulae I, II, III, IV, V,VI, VII, VIII, IX, X, XI, XII, and XIII, or a salt, solvate, hydrate, orprodrug thereof, and at least one pharmaceutically acceptable excipientto a subject in need thereof.

The invention includes use of a compound of the present invention in themanufacture of a medicament to prevent or treat a cell proliferationdisorder.

For example, the cell proliferation disorder is pre-cancer or cancer.The cell proliferation disorder treated or prevented by the compounds ofthe invention may be a cancer, such as, for example, colon cancer orlung cancer.

The cell proliferation disorder treated or prevented by the compounds ofthe invention may be a hyperproliferative disorder.

The cell proliferation disorder treated or prevented by the compounds ofthe invention may be psoriasis.

For example, the treatment or prevention of the proliferative disordermay occur through the inhibition of a tyrosine kinase. For example, thetyrosine kinase can be a Src kinase or focal adhesion kinase (FAK).

The invention relates to a method of treating or preventing a disease ordisorder that is modulated by tyrosine kinase inhibition, byadministering a pharmaceutical composition that includes a compoundaccording to Formula I or one of Formulae II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII, or a salt, solvate, hydrate, or prodrugthereof, and at least one pharmaceutically acceptable excipient. Forexample, the disease or disorder that is modulated by tyrosine kinaseinhibition is cancer, pre-cancer, a hyperproliferative disorder, or amicrobial infection. For example, the compound is a compound accordingto Formula I or II.

The invention relates to use of a compound of the present invention inthe manufacture of a medicament to treat or prevent a disease ordisorder that is modulated by tyrosine kinase inhibition.

The pharmaceutical composition of the invention may modulate a kinasepathway. For example, the kinase pathway is a Src kinase pathway, or afocal adhesion kinase pathway.

The pharmaceutical composition of the invention may modulate a kinasedirectly. For example, the kinase is Src kinase, or focal adhesionkinase.

Certain pharmaceutical compositions of the invention are non-ATPcompetitive kinase inhibitors.

The compounds of the invention are also useful to treat or prevent amicrobial infection, such as a bacterial, fungal, parasitic or viralinfection.

Certain pharmaceutical compositions of the invention include a compoundselected from Compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, and 137. For example, the pharmaceuticalcomposition includes Compound 33, 38, 40, 76, 133, 134, 136 or 137.

Certain pharmaceutical compositions of the invention include a compoundselected from Compound 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, and 247. For example, the pharmaceutical composition includesCompound 146 or 147.

Certain compounds of the invention are selected from Compounds 248-274.For example, the compound of the invention is Compound 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, or 274.

A compound of the invention may be used as a pharmaceutical agent. Forexample, a compound of the invention is used as an anti-proliferativeagent, for treating humans and/or animals, such as for treating humansand/or other mammals. The compounds may be used without limitation, forexample, as anti-cancer, anti-angiogenesis, anti-microbial,anti-bacterial, anti-fungal, anti-parasitic and/or anti-viral agents.Additionally, the compounds may be used for other cellproliferation-related disorders such as diabetic retinopathy, maculardegeneration and psoriases. Anti-cancer agents include anti-metastaticagents.

A compound of the present invention may be used in the manufacture of amedicament to be used as an anti-proliferative agent. A compound may beused in the manufacture of a medicament as an anti-cancer,anti-angiogenesis, anti-microbial, anti-bacterial, anti-fungal,anti-parasitic and/or anti-viral agent. Additionally, a compound may beused in the manufacture of a medicament for the treatment or preventionof other cell-proliferation-related disorders such as diabeticretinopathy, macular degeneration and psoriases.

The compound of the invention used as a pharmaceutical agent includes acompound selected from Compounds 1-136 and 137. For example, thecompound of the invention used as a pharmaceutical agent is Compound 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or137. For example, the compound of the invention used as a pharmaceuticalagent is selected from Compounds 33, 38, 40, 76, 133, 134, 136 and 137.

Certain pharmaceutical agents include a compound selected from thecompounds listed in Table 2. For example, the compound of the inventionused as a pharmaceutical agent is Compound 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171,172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213,214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227,228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241,242, 243, 244, 245, 246, or 247. For example, the compound of theinvention used as a pharmaceutical agent is 146 or 147.

Certain pharmaceutical agents include a compound selected from thecompounds listed in Table 3. For example, the compound of the inventionused as a pharmaceutical agent is Compound 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267,268, 269, 270, 271, 272, 273, or 274.

In one aspect of the invention, a compound of the invention, forexample, a compound of Formula I or one of Formulae II, III, IV, V, VI,VII, VIII, IX, X, XI, XII, and XIII, is used to modulate a kinasecascade. For example, the compound is used to modulate a component of akinase cascade which is responsible for the manifestation of a diseaseor disorder.

Such diseases and disorders include cancers, osteoporosis,cardiovascular disorders, immune system dysfunction, type II diabetes,obesity, and transplant rejection.

For example, a compound of the invention may be used to treat or preventa cell proliferation disorder in an subject. In one aspect of theembodiment, the cell proliferation disorder is pre-cancer or cancer. Inanother aspect of the embodiment, the cell proliferation disorder is ahyperproliferative disorder. In another embodiment, prevention ortreatment of the cell proliferation disorder, cancer orhyperproliferative disorder occurs through the inhibition of a kinase.In another embodiment, prevention or treatment of the cell proliferationdisorder, cancer or hyperproliferative disorder occurs through theinhibition of a tyrosine kinase. In another embodiment, prevention ortreatment of the cell proliferation disorder, cancer orhyperproliferative disorder occurs through the inhibition of Src kinaseor focal adhesion kinase (FAK). In another embodiment, the subject is amammal. In one embodiment, the subject is human.

The invention is also drawn to a method of treating or preventing canceror a proliferation disorder in a subject, comprising administering aneffective amount of a compound of the invention, for example, a compoundof Formula I or one of Formulae II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII. For example, the compound of the invention may be akinase inhibitor. The compound of the invention may be a non-ATPcompetitive kinase inhibitor. The compound of the invention may inhibita kinase directly, or it may affect the kinase pathway.

Another aspect of the invention includes compounds of Formula IA, andsalts, solvates, hydrates, or prodrugs thereof:

wherein: T is absent (i.e., the rings are connected by a bond), CR₁₂R₁₃,C(O), O, S, S(O), S(O)₂, NR₁₄, C(R₁₅R₁₆)C(R₁₇R₁₈), CH₂O, or OCH₂;

X_(y) is CZ, CY, N, or N—O;

X_(z) is CZ, CY, N, or N—O;

at least one of X_(y) and X_(z) is CZ;

Y is selected from hydrogen, hydroxyl, halogen, lower (C₁, C₂, C₃, C₄,C₅, or C₆) alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃,C₄, C₅, or C₆) alkyl-aryl, and O-benzyl;

X_(a) is CR_(a) or N, or N—O;

X_(b) is CR_(b), N, or N—O;

X_(c) is CR_(c) or N, or N—O;

X_(d) is CR_(d) or N, or N—O;

X_(e) is CR_(e), N, or N—O;

R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ are, independently,hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂,C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆)alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂-lower (C₁, C₂,C₃, C₄, C₅, or C₆) alkyl,

wherein W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅,or C₆ alkyl-aryl;

P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl is linear or branched alkyl;

K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, aryl,heteroaryl, or

L is aryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉,NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are independently C₁, C₂, C₃, C₄, C₅, or C₆ alkyl orR₁₉ and R₂₀ taken together with the attached nitrogen atom form a fivemembered ring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—;

R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently, H or C₁, C₂,C₃, C₄, C₅, or C₆ alkyl; and

Z is (CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, R₁, R₂, andR₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; and n and mare, independently 0, 1, or 2; provided that at least one of R_(a),R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ is P.

In one embodiment of the invention, at least one of X_(a), X_(b), X_(c),X_(d), X_(e), X_(y) and X_(z) is N. In another embodiment, at least twoof X_(a), X_(b), X_(c), X_(d), X_(e), X_(y) and X_(z) are N. In anotherembodiment, at least one of X_(a) and X_(y) is N. For example, bothX_(a) and X_(y) are N. In another embodiment, X_(a), X_(b), X_(c),X_(d), and X_(e) are not each N or N—O. In another embodiment, X_(c),X_(d), and X_(e) are not each N or N—O.

In one embodiment, T is absent. In another embodiment, X_(b) is CR_(b).In another embodiment, R_(b) is P. For example, in one embodiment, P isO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K. In one embodiment, lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl is CH₂CH₂CH₂. In one embodiment, lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl is branched alkyl. For example,branched alkyl is

In another embodiment, K, L, M, or Q, if present, is lower C₁, C₂, C₃,C₄, C₅ or C₆ alkoxy. For example, K is methoxy. In one embodiment,branched alkyl is

and K is methoxy. In another embodiment, K, L, M, or Q, if present, isCOOH. For example, in one embodiment, K is COOH. In another embodiment,K, L, M, or Q, if present, is aryl or heteroaryl. For example,heteroaryl is tetrazole.

In one embodiment, R_(b) is

In another embodiment, R_(b) is

In one embodiment, V is —OCH₂CH₂. In another embodiment, V is a bond. Inone embodiment, W is C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. For example, W ismethyl or ethyl.

In one embodiment, X_(z) is CZ, further wherein Z is

and R₇, R₈, R₉, R₁₀, and R₁₁ are selected from hydrogen, hydroxyl,halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁,C₂, C₃, C₄, C₅, or C₆ alkyl-OH, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-O—C₁,C₂, C₃, C₄, C₅, or C₆ alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl. In another embodiment, at least one of R₇, R₈, R₉, R₁₀,and R₁₁ is halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, or O-benzyl. Inanother embodiment, at least one of R₈ or R₁₀ is halogen. For example,halogen is fluorine. In another embodiment, at least one of R₇ or R₁₁ isC₁, C₂, C₃, C₄, C₅, or C₆ alkoxy or O-benzyl. For example, at least oneof R₇ or R₁₁ is ethoxy or at least one of R₇ or R₁₁ is O-benzyl. In oneembodiment, R₁₁ is H. In one embodiment, n is 1. In one embodiment, R₂is H. In one embodiment, R₃ is H. In one embodiment, m is 1. In anotherembodiment, m and n are each 1 and R₂ and R₃ are each H.

In one embodiment, R₄ and R₆ are each H. In another embodiment R₅ isselected from halogen and C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In oneembodiment, R₅ is halogen. For example, R₅ is Cl or F. In anotherembodiment, R₅ is C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. For example, R₅ ismethyl or ethyl.

The invention includes a solvate of a compound according to Formula IAor IB. The invention includes a hydrate of compound according to FormulaIA or IB. The invention includes an acid addition salt of a compoundaccording to Formula IA or IB. For example, a hydrochloride salt. Inanother embodiment, the invention includes a pharmaceutically acceptablesalt. The invention includes a composition comprising a compound ofFormula IA or IB and at least one pharmaceutically acceptable excipient.

Certain compounds of the invention include compounds selected fromCompound 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,and 274.

Another aspect of the invention includes a method of protecting againstor treating hearing loss in a subject comprising administering acompound having the Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII. In one embodiment, the compound inhibits oneor more components of a kinase signaling cascade. In one embodiment, thecompound is an allosteric inhibitor. In one embodiment, the compound isa peptide substrate inhibitor. In one embodiment, the compound does notinhibit ATP binding to the protein kinase. In one embodiment, thecompound inhibits a Src family protein kinase. In one embodiment, theSrc family protein kinase is pp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically e.g., by administering drops intothe ear, intraarterially, intralesionally, by metering pump, or byapplication to mucous membranes. In another embodiment, the compound isadministered with a pharmaceutically acceptable carrier.

In one embodiment, the compound is administered before initiation ofhearing loss. In another embodiment, the compound is administered afterinitiation of hearing loss.

In one embodiment, the compound is administered in combination with adrug that causes hearing loss e.g., cis platinum or an aminoglycosideantibiotic. In another embodiment, the compound is administered incombination with a drug that targets hairy cells.

In one embodiment, at least one of X_(a), X_(b), X_(c), X_(d), X_(e),X_(y) and X_(z) is N. In another embodiment, T is absent e.g., a bond.In another embodiment, X_(z) is CZ and Z is

In one embodiment, m and n are each 1 and R₂ and R₃ are each H. Inanother embodiment, at least one of R₇, R₈, R₉, R₁₀ and R₁₁ is halogen,C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, or O-benzyl. In one embodiment, thecompound is

Another aspect of the invention includes a method of protecting againstor treating osteoporosis in a subject comprising administering acompound having a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX,X, XI, XII, and XIII. In one embodiment, the compound inhibits one ormore components of a kinase signaling cascade. In another embodiment,the compound is an allosteric inhibitor. In one embodiment, the compoundis a peptide substrate inhibitor. In one embodiment, the compoundinhibits a Src family protein kinase. For example, the Src familyprotein kinase is pp60^(c-src) tyrosine kinase.

In one embodiment, at least one of X_(a), X_(b), X_(c), X_(d), X_(e),X_(y) and X_(z) is N. In another embodiment, T is absent e.g., a bond.In another embodiment, X_(z) is CZ and Z is

In one embodiment, m and n are each 1 and R₂ and R₃ are each H. Inanother embodiment, at least one of R₇, R₈, R₉, R₁₀ and R₁₁ is halogen,C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, or O-benzyl. In one embodiment, thecompound is Compound 25 (KX1-329), Compound 38 (KX2-377), Compound 76(KX2-361), Compound 133 (KX2-392), Compound 134 (KX2-391), or Compound137 (KX2-394).

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore initiation of osteoporosis. In another embodiment, the compoundis administered after initiation of osteoporosis.

Another aspect of the invention includes a method of protecting againstor treating ophthalmic diseases e.g., macular degeneration, retinopathy,macular edema, etc. in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. Another aspect of the invention includes use of acompound of the invention in the manufacture of a medicament to protectagainst or treat ophthalmic diseases. In one embodiment, the compoundinhibits one or more components of a kinase signaling cascade. Inanother embodiment, the compound is an allosteric inhibitor. In oneembodiment, the compound is a peptide substrate inhibitor. In oneembodiment, the compound inhibits a Src family protein kinase. Forexample, the Src family protein kinase is pp60^(c-src) tyrosine kinase.In another embodiment, the compound inhibits one or more components inthe VEGF pathway.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically (e.g., by administering drops tothe eye), intraarterially, intralesionally, by metering pump, or byapplication to mucous membranes. In one embodiment, the compound isadministered with a pharmaceutically acceptable carrier. In oneembodiment, the compound is administered before initiation of theophthalmic disease. In another embodiment, the compound is administeredafter initiation of ophthalmic disease.

Another aspect of the invention includes a method of protecting againstor treating diabetes in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XII. Another aspect of the invention includes use of a compoundof the invention in the manufacture of a medicament to protect againstor treat diabetes. In one embodiment, the compound inhibits one or morecomponents of a kinase signaling cascade. In another embodiment, thecompound is an allosteric inhibitor. In one embodiment, the compound isa peptide substrate inhibitor. In one embodiment, the compound inhibitsa Src family protein kinase. For example, the Src family protein kinaseis pp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore the onset of diabetes. In another embodiment, the compound isadministered after the onset of diabetes.

Another aspect of the invention includes a method of protecting againstor treating obesity in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. Another aspect of the invention includes use of acompound of the invention in the manufacture of a medicament to protectagainst or treat obesity. In one embodiment, the compound inhibits oneor more components of a kinase signaling cascade. In another embodiment,the compound is an allosteric inhibitor. In one embodiment, the compoundis a peptide substrate inhibitor. In one embodiment, the compoundinhibits a Src family protein kinase. For example, the Src familyprotein kinase is pp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore the subject is obese. In another embodiment, the compound isadministered after the subject is obese.

Another aspect of the invention includes a method of protecting againstor treating stroke in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. Another aspect of the invention includes use of acompound of the invention in the manufacture of a medicament to protectagainst or treat stroke. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore a stroke has occurred. In another embodiment, the compound isadministered after a stroke has occurred.

Another aspect of the invention includes a method of protecting againstor treating athrosclerosis in a subject comprising administering acompound having a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX,X, XI, XII, and XIII. Another aspect of the invention includes use of acompound of the invention in the manufacture of a medicament to protectagainst or treat athrosclerosis. In one embodiment, the compoundinhibits one or more components of a kinase signaling cascade. Inanother embodiment, the compound is an allosteric inhibitor. In oneembodiment, the compound is a peptide substrate inhibitor. In oneembodiment, the compound inhibits a Src family protein kinase. Forexample, the Src family protein kinase is pp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier.

Another aspect of the invention includes a method of regulating immunesystem activity in a subject comprising administering a compound havinga Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, andXIII. Another aspect of the invention includes use of a compound of theinvention in the manufacture of a medicament to regulate immune systemactivity. In one embodiment, the compound inhibits one or morecomponents of a kinase signaling cascade. In another embodiment, thecompound is an allosteric inhibitor. In one embodiment, the compound isa peptide substrate inhibitor. In one embodiment, the compound inhibitsa Src family protein kinase. For example, the Src family protein kinaseis pp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier.

Another aspect of the invention includes a method of protecting againstor treating chronic neuropathic pain in a subject comprisingadministering a compound having a Formulae I, IA, IB, II, III, IV, V,VI, VII, VIII, IX, X, XI, XII, and XIII. Another aspect of the inventionincludes use of a compound of the invention in the manufacture of amedicament to protect against or treat chronic neuropathic pain. In oneembodiment, the compound inhibits one or more components of a kinasesignaling cascade. In another embodiment, the compound is an allostericinhibitor. In one embodiment, the compound is a peptide substrateinhibitor. In one embodiment, the compound inhibits a Src family proteinkinase. For example, the Src family protein kinase is pp60^(c-src)tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore the onset of chronic neuropathic pain. In another embodiment, thecompound is administered after the onset of chronic neuropathic pain.

Another aspect of the invention includes a method of protecting againstor treating hepatitis B in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. Another aspect of the invention includes use of acompound of the invention in the manufacture of a medicament to protectagainst or treat hepatitis B. In one embodiment, the compound inhibitsone or more components of a kinase signaling cascade. In anotherembodiment, the compound is an allosteric inhibitor. In one embodiment,the compound is a peptide substrate inhibitor. In one embodiment, thecompound inhibits a Src family protein kinase. For example, the Srcfamily protein kinase is pp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore the onset of hepatitis B. In another embodiment, the compound isadministered after the onset of hepatitis B.

Another aspect of the invention is a method of preventing or treating acell proliferation disorder comprising administering to a subject inneed thereof a compound having the Formula IA. Another aspect of theinvention includes use of a compound of the invention in the manufactureof a medicament to protect against or treat a cell proliferationdisorder. In one embodiment, the compound inhibits one or morecomponents of a protein kinase signaling cascade. In another embodiment,the compound is an allosteric inhibitor. In another embodiment, thecompound is a peptide substrate inhibitor. In another embodiment, thecompound does not inhibit ATP binding to a protein kinase. In oneembodiment, the compound inhibits a Src family protein kinase. Inanother embodiment, the Src family protein kinase is pp60^(c-src)tyrosine kinase.

In one embodiment, at least one of X_(a), X_(b), X_(c), X_(d), X_(e),X_(y) and X_(z) is N. In another embodiment, X_(z) is CZ, furtherwherein Z is

and R₇, R₈, R₉, R₁₀, and R₁₁ are selected from hydrogen, hydroxyl,halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁,C₂, C₃, C₄, C₅, or C₆ alkyl-OH, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-O—C₁,C₂, C₃, C₄, C₅, or C₆ alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl. In one embodiment, at least one of R₇, R₈, R₉, R₁₀, andR₁₁ is halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, or O-benzyl. Inanother embodiment, m and n are each 1 and R₂ and R₃ are each H. In oneembodiment, R₄ and R₆ are each H. In one embodiment of the invention, acompound is selected from 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, or 274.

Accordingly, another aspect of the invention is a method for treatingleukemia in a host comprising administering to a patient a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. Another aspect of the invention includes use of acompound of the invention in the manufacture of a medicament to treatleukemia.

In another embodiment, there is provided a method for treating leukemiain a host comprising administering to a patient a therapeuticallyeffective amount of a compound according to Formulae I, IA, IB, II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII, as defined above, and atleast one further therapeutic agent selected from the group consistingof anti-proliferative agents, cytotoxic agents, cytostatic agents, andchemotherapeutic agents and salts and derivatives thereof. According tocertain embodiments, the compound of the present invention may be usedin the treatment of a leukemia in combination therapy with one or moreof the drugs selected from a group consisting of an alkaloid, analkylating agent, an antitumor antibiotic, an antimetabolite, an Bcr-Abltyrosine kinase inhibitor, a nucleoside analog, a multidrug resistancereversing agent, a DNA binding agent, microtubule binding drug, a toxinand a DNA antagonist. Those of skill in the art will recognize thechemotherapeutic agents classified into one or more particular classesof drugs described above.

In one embodiment, there is provide a method for treating leukemia in ahost comprising administering to a patient that has been previouslytreated with a Bcr-Abl tyrosine kinase inhibitor and has becomeresistant to the Bcr-Abl tyrosine kinase inhibitor treatment, atherapeutically effective amount of a compound according to Formulae I,IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII.

The above description sets forth rather broadly the more importantfeatures of the present invention in order that the detailed descriptionthereof that follows may be understood, and in order that the presentcontributions to the art may be better appreciated. Other objects andfeatures of the present invention will become apparent from thefollowing detailed description considered in conjunction with theexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph indicating the effect of AZ28 and KX2-391 on Srcautophosphorylation in c-Src/NIH-3T3 cells; FIG. 1B is a graphindicating the effect of AZ28 and KX2-391 on Src autophosphorylation inHT-29 cells.

FIG. 2A is a graph indicating the effect of AZ28 and KX2-391 on FAKphosphorylation in c-Src/NIH-3T3 cells; FIG. 2B is a graph indicatingthe effect of AZ28 and KX2-391 on FAK phosphorylation in HT-29 cells.

FIG. 3A is a graph indicating the effect of AZ28 and KX2-391 on Shcphosphorylation in c-Src/NIH-3T3 cells; FIG. 3B is a graph indicatingthe effect of AZ28 and KX2-391 on Shc phosphorylation in HT-29 cells.

FIG. 4 is a graph indicating the effect of AZ28 and KX2-391 on paxillinphosphorylation in c-Src/NIH-3T3 cells.

FIG. 5A is a graph indicating the effect of AZ28 and KX2-391 oncaspase-3 cleavage in c-Src/NIH-3T3 cells; FIG. 5B is a graph indicatingthe effect of AZ28 and KX2-391 on caspase-3 cleavage in HT-29 cells.

FIG. 6A is a graph indicating the effect of AZ28 and KX2-391 on totalphosphotyrosine levels in c-Src/NIH-3T3 cells; FIG. 6B is a graphindicating the effect of AZ28 and KX2-391 on total phosphotyrosinelevels in HT-29 cells.

FIG. 7 is a graph indicating the effect of AZ28 and KX2-391 onautophosphorylation of PDGFR in c-Src/NIH-3T3 cells.

FIG. 8A is a graph indicating the effect of AZ28 and KX2-391 onautophosphorylation of FAK in c-Src/NIH-3T3 cells; FIG. 8B is a graphindicating the effect of AZ28 and KX2-391 on autophosphorylation of FAKin HT-29 cells.

FIG. 9A is a graph indicating the effect of AZ28 and KX2-391 onautophosphorylation of EGFR in c-Src/NIH-3T3 cells; FIG. 9B is a graphindicating the effect of AZ28 and KX2-391 on autophosphorylation of EGFRin HT-29 cells.

FIG. 10 is a bar chart showing the average threshold shifts (dB) inchinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and8 kHz band noise on day 1 after experimental manipulation.

FIG. 11 is a graph showing the average threshold shifts (dB) inchinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and8 kHz band noise on day 7 after experimental manipulation.

FIG. 12 is a graph showing the average threshold shifts (dB) inchinchilla cochleas after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz, and8 kHz band noise on day 21 after experimental manipulation.

FIG. 13 is a line graph showing the threshold shifts (dB) in guinea pigcochleas after exposure to 2 kHz, 4 kHz, 8 kHz, 12 kHz, 16 kHz and 20kHz band noise after treatment with cisplatin.

FIG. 14 is a line graph showing the threshold shifts (dB) inKX1-004-treated guinea pig cochleas after exposure to 2 kHz, 4 kHz, 8kHz, 12 kHz, 16 kHz and 20 kHz band noise after treatment withcisplatin.

FIG. 15 is a line graph showing the average threshold shifts (dB) inKX1-004-treated guinea pig cochleas and untreated control guinea pigcochleas after exposure to 2 kHz, 4 kHz, 8 kHz, 12 kHz, 16 kHz and 20kHz band noise after treatment with cisplatin.

FIG. 16A shows a comparison of taxol and doxorubicin (they were moreeffective than etoposide and cisplatin in this tumor cell culture) withthe three Src inhibitors (45, 43-meta, and 49-meta from Table V)utilizing ovarian tumor cells from tumor N015.

FIG. 16B shows the results from tests of the Src inhibitors forinhibition of normal human fibroblast cell growth. No inhibition ofnormal cell growth (both subconfluent and confluent; some enhancedgrowth was observed instead) was found, indicating that these inhibitorsare not toxic to normal cells even at a 10-fold higher concentration.FIG. 16C shows the results from tests of two of the Src inhibitors forinhibition of is v-Src stimulated LA25 cell growth. FIG. 16D shows theresults from tests of two of the Src inhibitors for inhibition of normalrat kidney cell growth. FIG. 16E provides the structures of the Srcinhibitors 45, 43-meta, and 49-meta.

FIG. 16 is a series of illustrations depicting the effect of compoundson osteoclast formation.

FIG. 17 is a bar chart demonstrating the effect of compounds onosteoclast formation.

FIG. 18 is a series of illustrations showing the effect of compounds onosteoclast survival.

FIG. 19 is a bar chart depicting the effect of compounds on osteoclastsurvival.

FIG. 20A is a bar chart demonstrating the effect of compounds on boneresorption in vitro.

FIG. 20B is a bar chart showing the effect of compounds on resorptionpit formation.

FIG. 21A is a series of illustrations depicting the effect of compoundson osteoclast formation on bone slices.

FIG. 21B is a series of illustrations demonstrating the effect ofcompounds on the formation of resorption pits on bone slices.

FIG. 22 is a bar chart showing the effect of compounds on alkalinephosphatase expression by osteoblasts.

FIG. 23 is a bar chart depicting the effect of compounds on proteinexpression by osteoblasts.

FIG. 24 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in COLO-320DM cells. Data was normalized torepresent percent of maximum response. Dasatinib is BMS-354825.

FIG. 25 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in COLO-320DM cells. Data is provided in pureOD₅₇₀ signal format.

FIG. 26 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in H460 cells. Data was normalized to representpercent of maximum response.

FIG. 27 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in H460 cells. Data is provided in pure OD₅₇₀signal format.

FIG. 28 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in H226 cells. Data was normalized to representpercent of maximum response.

FIG. 29 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in H226 cells. Data is provided in pure OD₅₇₀signal format.

FIG. 30 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in HCT-116 cells. Data was normalized torepresent percent of maximum response.

FIG. 31 shows growth inhibition curves and EC₅₀/IC₅₀ for Compound 134(KX2-391) and Dasatinib in HCT-116 cells. Data is provided in pure OD₅₇₀signal format.

FIG. 32 is a graph indicating the effect of Dasatinib on Dasatinib andImatinib resistant leukemia cells.

FIG. 33 is a graph indicating the effect of compound 134 (KX2-391/KXO1)on Dasatinib and Imatinib resistant leukemia cells.

FIG. 34 shows the growth inhibition curves and GI₅₀ of the combinationof Gemzar and Compound 134 (KXO1/KX2-391) in the L3.6pl cell line usingthe BrdU assay.

FIG. 35 shows the growth inhibition curves and GI₅₀ of Gemzar andCompound 134 (KXO1/KX2-391) in the L3.6pl cell line using the BrdUassay.

FIG. 36 shows the growth inhibition curves and GI₅₀ of Compound 134(KXO1/KX2-391) as compared to Dasatinib (BMS354825) in HT-29 cells.

FIG. 37 shows the growth inhibition curves and GI₅₀ of Compound 134(KXO1/KX2-391) as compared to Dasatinib (BMS354825) in SKOV-3 cells.

FIG. 38 shows the growth inhibition curves and GI₅₀ of Compound 134(KXO1/KX2-391) as compared to Dasatinib (BMS354825) in A549 cells.

FIG. 39 shows the growth inhibition curves and GI₅₀ of Compound 134(KXO1/KX2-391) as compared to Dasatinib (BMS354825) in K562 cells.

FIG. 40 shows the growth inhibition curves and GI₅₀ of Compound 134(KXO1/KX2-391) as compared to Dasatinib (BMS354825) in MDA-MB-231 cells.

FIG. 41 shows the tumor weight from the orthotopic prostate model formeasuring in vivo metastases at various concentration of Compound 134(KXO1/KX2-391).

FIG. 42 is a bar graph of the screening results for Anti-HBV efficacyand cellular cytotoxicity.

FIGS. 43A and 43B are a series of graphs depicting the inhibition of theisolated kinases Src (FIG. 43A) and FAK (FIG. 43B) by the KXO1 compound.KXO1 weakly inhibited isolated Src (IC50=46 μM).

FIGS. 44A, 44B, 44C, and 44D are a series of graphs depicting theinhibition of Src kinase activity in whole cells. FIG. 44A is a graphdepicting the effect of KXO1 on Src autophosphorylation in c-Src/NIH-3T3cells; FIG. 44B is a graph indicating the effect of KXO1 on Srcautophosphorylation in HT-29 cells; FIG. 44C is a graph depicting theeffect of KXO1 on Src transphosphorylation in c-Src/NIH-3T3 cells; andFIG. 44D is a graph indicating the effect of KXO1 on Srcautophosphorylation in HT-29 cells.

FIG. 45 is an illustration depicting the selectivity of KXO1 for proteintyrosine kinases (PTKs) in whole cells as compared to Dasatinib, anATP-competitive Src inhibitor currently in clinical trials.

FIG. 46 is a graph depicting the oral potency of KXO1 in mousexenografts. KXO1 demonstrated higher oral potency in staged HT29 (ahuman colon cancer cell line) mouse than Dasatinib.

FIG. 47 shows a second week IVIS follow up study after the treatment ofKXO1 at 2.5 mg/dose bid, 5.0 mg/dose bid, and Dasatinib 7.5 mg/dose bid.

FIG. 48 is a graph showing the survival of C57BL/6 mice bearingintracranial GL261 gliomas over a 40 day period.

FIGS. 49A-F are a series of graphs showing the weight gain in each ofthe C₅₇BL/6 mice in the different treatment groups of the intracranialGL261 glioma survival study.

FIG. 50 is a graph showing the average weights over a 40-day period foreach of the treatment groups in the intracranial GL261 glioma survivalstudy.

FIG. 51 is a graph showing synergistic growth inhibitory effects oftamoxifen and KXO1 on MCF-7 cells.

DETAILED DESCRIPTION OF THE INVENTION

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description. In thespecification, the singular forms also include the plural unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control.

Because kinases are involved in the regulation of a wide variety ofnormal cellular signal transduction pathways (e.g., cell growth,differentiation, survival, adhesion, migration, etc.), kinases arethought to play a role in a variety of diseases and disorders. Thus,modulation of kinase signaling cascades may be an important way to treator prevent such diseases and disorders. Such diseases and disordersinclude, for example, cancers, osteoporosis, cardiovascular disorders,immune system dysfunction, type II diabetes, obesity, and transplantrejection.

Compounds of the invention are useful in modulation a component of thekinase signaling cascade. Some compounds may be useful in modulation ofmore than one component of a kinase signaling cascade. The phrase“modulates one or more components of a protein kinase signaling cascade”means that one or more components of the kinase signaling cascade areaffected such that the functioning of a cell changes. Components of aprotein kinase signaling cascade include any proteins involved directlyor indirectly in the kinase signaling pathway including secondmessengers and upstream and downstream targets.

A number of protein kinases and phosphatases are known, and are targetsfor the development of therapeutics. See, e.g., Hidaka and Kobayashi,Annu. Rev. Pharmacol. Toxicol, 1992, 32:377-397; Davies et al., Biochem.J., 2000, 351:95-105, each of which is incorporated by reference herein.

One family of kinases, the protein tyrosine kinases are divided into twolarge families: receptor tyrosine kinases, or RTKs (e.g., insulinreceptor kinase (IRK), epidermal growth factor receptor (EGFR), basicfibroblast growth factor receptor (FGFR), platelet-derived growth factorreceptor (PDGFR), vascular endothelial growth factor receptor (VEGFR-2or Flk1/KDR), and nerve growth factor receptor (NGFR)) and nonreceptortyrosine kinases, or NRTKs (e.g., the Src family (Src, Fyn, Yes, Blk,Yrk, Fgr, Hck, Lck, and Lyn), Fak, Jak, Abl and Zap70). See, forexample, Parang and Sun, Expert Opin. Ther. Patents, 2005, 15:1183-1207,incorporated by reference herein.

Because of the role of Src kinases in a variety of cancers, thesekinases are the subject of a number of studies relating to thedevelopment of Src inhibitors as cancer therapeutics, including highlymetastatic cancer cell growth. Src inhibitors are sought as therapeuticsfor a variety of cancers, including, for example, colon cancer,precancerous colon lesions, ovarian cancer, breast cancer, epithelialcancers, esophageal cancer, non-small cell lung cancer, pancreaticcancer, and others. See, e.g., Frame, Biochim. Biophys. Acta, 2002,1602:114-130 and Parang and Sun, Expert Opin. Ther. Patents, 2005,15:1183-1207.

Inhibition of other kinases may be useful in the treatment andmodulation of other types of diseases and disorders. For example,various eye diseases may be inhibited or prevented by administration ofVEGF receptor tyrosine kinase inhibitors. Inhibitors of the tyrosinephosphatase PTP-1B and/or glycogen phosphorylase may provide treatmentsfor Type II diabetes or obesity. Inhibitors of p56lck may be useful intreating immune system disorders. Other targets include HIV reversetranscriptase, thromboxane synthase, EGFRTK, p55 fyn, etc.

Compounds of the invention may be Src signaling inhibitors that bind inthe Src peptide substrate site. The activity of various compounds of theinvention has been studied in c-Src (527F, constitutively active andtransforming) transformed NIH3T3 cells and in human colon cancer cells(HT29). For example, in these cell lines, KX2-391 was shown to reducethe phosphorylation level of known Src protein substrates in adose-dependent fashion and in good correlation with growth inhibitoryeffects. Thus, in some embodiments, compounds of the invention maydirectly inhibit Src, and may do so by binding in the peptide bindingsite (as opposed to binding at an allosteric site).

Molecular modeling experiments have been performed which show thatcompounds of the invention fit into the model Src substrate site (See,e.g., U.S. Pat. Nos. 7,005,445 and 7,070,936). Modeling is also used toretool the Src kinase inhibitor scaffolds in order to target otherkinases, simply by using a different set of side chains present on themolecules and/or modifying the scaffold itself.

Without wishing to be bound by theory, it is believed that theconformation of some kinases (e.g., Src) outside cells relative to theconformation inside cells is markedly different, because inside cells,many kinases are is embedded in multiprotein signaling complexes. Thus,because the peptide substrate binding site is not well formed in anisolated kinase (as shown by Src x-ray structures), it is believed thatthe activity against isolated kinase for a peptide substrate bindinginhibitor would be weak. Binding to this site in an isolated kinaseassay requires the inhibitor to capture the very small percentage oftotal protein in an isolated enzyme assay that is in the sameconformation that exists inside cells. This requires a large excess ofthe inhibitor to drain significant amounts of the enzyme from thecatalytic cycle in the assay in order to be detectable.

However, for cell-based assays, a large inhibitor excess is not neededbecause the peptide binding site is expected to be formed. In cell-basedSrc assays, SH2 & SH3 domain binding proteins have already shifted theSrc conformation so that the peptide substrate binding site is fullyformed. Thus, low concentrations of the inhibitor can remove the enzymefrom the catalytic cycle since all of the enzyme is in the tight bindingconformation.

The vast majority of known kinase inhibitors are ATP competitive andshow poor selectivity in a panel of isolated kinase assays. However,many of the compounds of the invention are thought to be peptidesubstrate binding inhibitors. Thus, traditional high throughputscreening of compounds against isolated enzymes, such as Src, would notresult in the discovery of compounds of the invention.

There is considerable recent literature support for targeting pp60c-src(Src) as a broadly useful approach to cancer therapy without resultingin serious toxicity. For example, tumors that display enhanced EGFreceptor PTK signaling, or overexpress the related Her-2/neu receptor,have constitutively activated Src and enhanced tumor invasiveness.Inhibition of Src in these cells induces growth arrest, triggersapoptosis, and reverses the transformed phenotype (Karni et al. (1999)Oncogene 18(33): 4654-4662). It is known that abnormally elevated Srcactivity allows transformed cells to grow in an anchorage-independentfashion. This is apparently caused by the fact that extracellular matrixsignaling elevates Src activity in the FAK/Src pathway, in a coordinatedfashion with mitogenic signaling, and thereby blocks an apoptoticmechanism which would normally have been activated. Consequently FAK/Srcinhibition in tumor cells may induce apoptosis because the apoptoticmechanism which would have normally become activated upon breaking freefrom the extracellular matrix would be induced (Hisano, et al., Proc.Annu. Meet. Am. Assoc. Cancer Res. 38:A1925 (1997)). Additionally,reduced VEGF mRNA expression was noted upon Src inhibition and tumorsderived from these Src-inhibited cell lines showed reduced angiogenicdevelopment (Ellis et al., Journal of Biological Chemistry 273(2):1052-1057 (1998)).

For example, a knock-out of the Src gene in mice led to only one defect,namely osteoclasts that fail to form ruffled borders and consequently donot resorb bone. However, the osteoclast bone resorb function wasrescued in these mice by inserting a kinase defective Src gene(Schwartzberg et al., (1997) Genes & Development 11: 2835-2844). Thissuggested that Src kinase activity can be inhibited in vivo withouttriggering the only known toxicity because the presence of the Srcprotein is apparently sufficient to recruit and activate other PTKs(which are essential for maintaining osteoclast function) in anosteoclast essential signaling complex.

Src has been proposed to be a “universal” target for cancer therapysince it has been found to be overactivated in a growing number of humantumors (Levitzki, Current Opinion in Cell Biology, 8, 239-244 (1996);Levitzki, Anti-Cancer Drug Design, 11, 175-182 (1996)). The potentialbenefits of Src inhibition for cancer therapy appear to be four-foldinhibition of uncontrolled cell growth caused by autocrine growth factorloop effects, inhibition of metastasis due to triggering apoptosis uponbreaking free from the cell matrix, inhibition of tumor angiogenesis viareduced VEGF levels, low toxicity.

Prostate cancer cells have been reported to have both an over expressionof paxillin and p130cas and are hyperphosphorylated (Tremblay et al.,Int. J. Cancer, 68, 164-171, 1996) and may thus be a prime target forSrc inhibitors.

Thus, the invention relates to compounds and methods of using compoundsto treat cell proliferation disorders.

In one embodiment, a compound of the invention may be used to treat orprevent brain cancer in a subject. Another aspect of the inventionincludes use of a compound of the invention in the manufacture of amedicament to treat or prevent brain cancer. In order to protect againstbrain cancer, the compound may be administered prior to the developmentof brain cancer in a subject. Alternatively, the compound may be used totreat brain cancer in a subject. A compound of the instant inventionused to treat or prevent brain cancer may be involved in modulating akinase signaling cascade e.g., a kinase inhibitor, a non-ATP competitiveinhibitor, a tyrosine kinase inhibitor, a protein kinase phosphataseinhibitor or a protein-tyrosine phosphates 1B inhibitor.

The term “brain cancer” encompasses a variety of cancers. There can beactual brain tumors which arise from the brain itself, known as primarybrain cancers of which there are several. The term “brain cancer” refersto malignant tumors i.e., tumors that grow and spread aggressively,overpowering healthy cells by taking up their space, blood, andnutrients. Tumors that do not spread aggressively are called benigntumors. Benign tumors are generally less serious than a malignant tumor,but a benign tumor can still cause problems in the brain. There can alsobe brain metastases, which represent the spread of other cancers, suchas lung or breast to the brain.

Brain tumors are classified by both the cell of the brain that makesthem up and how the tumor looks under the microscope. Primary braintumors arise from any of the cells in the brain, or from specificstructures in the brain. Glia cells support the neurons of the brain andtumors which arise from these cells are known as glial tumors. Themembrane that surrounds the brain can also develop tumors and these areknown as meningiomas. There are other types of tumors, which involveother structures of the brain including ependymoma. The most commonprimary brain tumors are gliomas, meningiomas, pituitary adenomas,vestibular schwannomas, and primitive neuroectodermal tumors(medullablastomas).

The present invention provides a method of treating or preventingglioblastoma, a malignant rapidly growing astrocytoma of the centralnervous system and usually of a cerebral hemisphere. Synonyms forglioblastoma include glioblastoma multiforme (GBM), giant cellglioblastoma, and multiforme spongioblastoma multiforme. Gioblastoma isthe most common malignant primary brain tumor and has proven verydifficult to treat. These tumors are often aggressive and infiltratesurrounding brain tissue. Glioblastomas arise from glial cells, whichare cells that form the tissue that surrounds and protects other nervecells found within the brain and spinal cord. Gioblastomas are mainlycomposed of star-shaped glial cells known as astrocytes. The term“glioma” includes any type of brain tumor such as astrocytomas,oligodendrogliomas, ependymomas, and choroid plexus papillomas.Astrocytomas come in four grades based on how fast the cells arereproducing and the likelihood that they will infiltrate nearby tissue.Grades I or II astrocytomas are nonmalignant and may be referred to aslow-grade. Grades III and IV astrocytomas are malignant and may bereferred to as high-grade astrocytomas. Grade II astrocytomas are knownas anaplastic astrocytomas. Grade IV astrocytomas are known asglioblastoma multiforme.

The invention provides a method of treating or preventingmedulloblastoma. Medulloblastoma is a highly malignant primary braintumor that originates in the cerebellum or posterior fossa. Originallyconsidered to be a glioma, medulloblastoma is now known to be of thefamily of cranial primitive neuroectodermal tumors (PNET).

Tumors that originate in the cerebellum are referred to asinfratentorial because they occur below the tentorium, a thick membranethat separates the cerebral hemispheres of the brain from thecerebellum. Another term for medulloblastoma is infratentorial PNET.Medulloblastoma is the most common PNET originating in the brain. AllPNET tumors of the brain are invasive and rapidly growing tumors that,unlike most brain tumors, spread through the cerebrospinal fluid (CSF)and frequently metastasize to different locations in the brain andspine. The peak of occurrence of medullablastoma is seven years of age.Seventy percent of medulloblastomas occur in individuals younger than16. Desmoplastic medulloblastoma is encountered especially in adulthood.This type of tumor rarely occurs beyond the fifth decade of life.

The present invention provides a method for treating or preventingneuroblastoma, a cancer that forms in nerve tissue. The cells ofneuroblastoma usually resemble very primitive developing nerve cellsfound in an embryo or fetus. The term neuro indicates “nerves,” whileblastoma refers to a cancer that affects immature or developing cells.Neurons (nerve cells) are the main component of the brain and spinalcord and of the nerves that connect them to the rest of the body.Neuroblastoma usually begins in the adrenal glands, but it may alsobegin in the spinal cord. Neuroblastoma is the most common extracranialsolid cancer in childhood. In 2007, neuroblasoma was the most commoncancer in infancy, with an annual incidence of about 650 new cases peryear in the US. Close to 50 percent of neuroblastoma cases occur inchildren younger than two years old. It is a neuroendocrine tumor,arising from any neural crest element of the sympathetic nervous systemor SNS. A branch of the autonomic nervous system, the SNS is a nervenetwork that carries messages from the brain throughout the body and isresponsible for the fight-or-flight response and production ofadrenaline or epinephrine.

The invention provides a method of treating or preventingneuroepithelioma, malignant tumors of the neuroepithelium.Neuroepithelioma is found most commonly in children and young adults. Itarises most often in the chest wall, pelvis, or extremity, either inbone or soft tissue. Procedures used in the diagnosis may include bloodand urine tests, X rays of the affected bone and the whole body andlungs, bone marrow aspirations, CT scans, and fluoroscopy. Treatmentsinclude surgery, radiation therapy and chemotherapy. Ewing's tumors arean example of a type of peripheral neuroepithelioma.

Kinases have been shown to play a role in brain cancers. Gene expressionprofiles of glioblastoma multiforme have identified tyrosine kinases asplaying a role in glioma migration/invasion. For example, PYK2 is amember of the focal adhesion family of nonreceptor tyrosine kinases; itis closely involved with src-induced increased actin polymerization atthe fibroblastic cell periphery. Its role in glioma migration/invasionhas become more clear, as overexpression of PYK2 induced glioblastomacell migration in culture. Levels of activated PYK2 positivelycorrelated with the migration phenotype in four glioblastoma cell lines(SF767, G112, T98G and U118). Analysis of activated PYK2 in GBMinvastion in situ revealed strong staining in infiltrating GBM cells.(See, Hoelzinger et al, Neoplasia, vol. 7 (1)7-16. Thus, modulation of akinase receptor using a compound of the invention may be useful in theprevention or treatment of brain cancers such as glioblastomamultiforme.

Alternatively, a compound of the invention may be used to treat orprevent renal cancer in a subject. Another aspect of the inventionincludes use of a compound of the invention in the manufacture of amedicament to treat or prevent renal cancer. In order to protect againstrenal cancer, the compound may be administered prior to the developmentof renal cancer in a subject. Alternatively, the compound may be used totreat renal cancer in a subject. A compound of the instant inventionused to treat or prevent renal cancer may be involved in modulating akinase signaling cascade e.g., a kinase inhibitor, a non-ATP competitiveinhibitor, a tyrosine kinase inhibitor, a protein kinase phosphataseinhibitor or a protein-tyrosine phosphates 1B inhibitor.

Several types of cancer can develop in the kidneys. Renal cell carcinoma(RCC), the most common form, accounts for approximately 85% of allcases. The present invention provides a method of treating or preventingrenal cell carcinoma. The invention also provides a method for thetreatment of other types of kidney cancer including, for example, renalpelvis carcinoma (cancer that forms in the center of the kidney whereurine collects), Wilms tumors, which are a type of kidney cancer thatusually develops in children under the age of 5, clear cell carcinomaalso called clear cell adenocarcinoma and mesonephroma (a tumor type,usually of the female genital tract, in which the inside of the cellslook clear when viewed under a microscope), renal adenocarcinoma (a typeof kidney tumor characterized by the development of finger-likeprojections in at least some of the tumor), and renal rhabdomyosarcoma,a rare and highly aggressive tumor in the adult population.

In RCC, cancerous (malignant) cells develop in the lining of thekidney's tubules and grow into a tumor mass. In most cases, a singletumor develops, although more than one tumor can develop within one orboth kidneys. RCC is characterized by a lack of early warning signs,diverse clinical manifestations, resistance to radiation andchemotherapy, and infrequent but reproducible responses to immunotherapyagents such as interferon alpha and interleukin (IL)-2. In the past, RCCtumors were believed to derive from the adrenal gland; therefore, theterm hypernephroma was used often.

The tissue of origin for renal cell carcinoma is the proximal renaltubular epithelium. Renal cancer occurs in both a sporadic(nonhereditary) and a hereditary form, and both forms are associatedwith structural alterations of the short arm of chromosome 3 (3p).Genetic studies of the families at high risk for developing renal cancerled to the cloning of genes whose alteration results in tumor formation.These genes are either tumor suppressors (VHL, TSC) or oncogenes (MET).At least 4 hereditary syndromes associated with renal cell carcinoma arerecognized: (1) von Hippel-Lindau (VHL) syndrome, (2) hereditarypapillary renal carcinoma (HPRC), (3) familial renal oncocytoma (FRO)associated with Birt-Hogg-Dube syndrome (BHDS), and (4) hereditary renalcarcinoma (HRC).

RCC has a very poor prognosis, mainly because, in nearly 30% of allpatients with localized disease, 40% of them develop distant metastasesfollowing removal of the primary tumor. The age-adjusted incidence ofrenal cell carcinoma has been rising by 3% per year. According to theAmerican Cancer Society, in 2007 there were approximately 51,500 casesof malignant tumors of the kidney diagnosed in the United States withapproximately 12,500 deaths; renal cell cancer accounted for 80% of thisincidence and mortality. Radical nephrectomy is the main treatment forlocalized RCC. However radiotherapy and available chemotherapeuticagents are ineffective against advanced and metastic RCC.

Immunotherapy using interferon-α and interluckin-2 is effective in onlya small percentage of patients with metastatic RCC and is extremelytoxic. Recently, kinase inhibitors have been developed for the treatmentof renal cancer e.g., Gleevec® and other new agents, such as sorafeniband sunitinib, having anti-angiogenic effects through targeting multiplereceptor kinases, have shown activity in patients failing immunotherapy.However, these treatments are also not without limitations. For example,it's been found that the effect of Gleevec® is limited to a certain typeof tumor and resistance can develop. Also, it is recommended thatpatients taking sunitinib should be monitored for cardiovascular sideeffects such as hypertension. As such, a need exists for the developmentof methods for the treatment and prevention of renal cancer.

Alternatively, a compound of the invention may be used to treat orprevent liver cancer in a subject. Another aspect of the inventionincludes use of a compound of the invention in the manufacture of amedicament to treat or prevent liver cancer. In order to protect againstliver cancer, the compound may be administered prior to the developmentof liver cancer in a subject. Alternatively, the compound may be used totreat liver cancer in a subject. A compound of the instant inventionused to treat or prevent liver cancer may be involved in modulating akinase signaling cascade e.g., a kinase inhibitor, a non-ATP competitiveinhibitor, a tyrosine kinase inhibitor, a protein kinase phosphataseinhibitor or a protein-tyrosine phosphates 1B inhibitor.

Several types of cancer can develop in the liver. Hepatocellularcarcinoma (HCC) accounts for 80-90% of all liver cancers. The presentinvention provides a method of treating or preventing hepatocellularcarcinoma. HCC begins in the hepatocytes, the main type of liver cell.About 3 out of 4 primary liver cancers are this type. HCC can havedifferent growth patterns. Some begin as a single tumor that growslarger. Only late in the disease does it spread to other parts of theliver. HCC may also begin in many spots throughout the liver and not asa single tumor.

The invention also provides a method for the treatment of other types ofliver cancer including, for example, cholangiocarcinomas, which startsin the bile ducts of the gallbladder; angiosarcomas and hemangiosarcomasare two other forms of cancer that begin in the blood vessels of theliver. These tumors grow quickly. Often by the time they are found theyare too widespread to be removed and treatment may not help very much;hepatoblastoma is a cancer that develops in children, usually found inchildren younger than 4 years old.

Kinases have been shown to play a role in liver cancer. For example,changes known to occur in human HCC are overexpression, amplification ormutation of the protooncogene MET, which encodes the receptor proteintyrosine kinase Met (See, Tward et al., PNAS, vol. 104 (37)14771-14776).It's also been demonstrated that FAK is involved in early events ofintegrin-mediated adhesion of circulating carcinoma cells under fluidflow in vitro and in vivo. It is thought that this kinase may take partin the establishment of definite adhesion interactions that enableadherent tumor cells to resist shear forces (See, Sengbusch et al.,American Journal of Pathology, vol 166 (2)585-595). In 2007, livercancer was the third leading cause of cancer-related deaths worldwide,and the sixth most widespread cancer globally. 600,000 people areannually are diagnosed with liver cancer worldwide and the incidence isrising. Accordingly, a need exists for the development of methods forthe treatment and prevention of liver cancer.

The compounds of the present invention are useful as pharmaceuticalagents, for example, as therapeutic agents for treating humans andanimals. The compounds may be used without limitation, for example, asanti-cancer, anti-angiogenesis, anti-metastatic, anti-microbial,anti-bacterial, anti-fungal, anti-parasitic and/or anti-viral agents.The compounds may be used for other cell proliferation-related disorderssuch as psoriases.

As described herein, a compound of the invention may be used to protectagainst or prevent hearing loss in a subject. Another aspect of theinvention includes use of a compound of the invention in the manufactureof a medicament to protect against or treat hearing loss. In order toprotect against hearing loss, the compound may be administered prior tonoise exposure or exposure to a drug which induces hearing loss. Suchdrugs may include chemotherapeutic drugs (e.g., platinum-based drugswhich target hair cells) and aminoglycoside antibiotics. A compound ofthe invention may provide a synergistic effect with certain cancerdrugs. For example, promising inhibitors can be screened in primaryhuman tumor tissue assays, particularly to look for synergy with otherknown anti-cancer drugs. In addition, the protein kinase inhibitors mayreduce toxicity of certain cancer drugs (e.g., platinum-based drugswhich are toxic to the cochlea and kidney), thereby allowing increaseddosage.

Alternatively, a compound of the invention may be used to treat hearingloss in a subject. In this embodiment, the compound is administered tothe subject subsequent to the initiation of hearing loss to reduce thelevel of hearing loss. A compound of the invention may be involved inmodulating a kinase cascade, e.g. a kinase inhibitor, a non-ATPcompetitive inhibitor, a tyrosine kinase inhibitor, a Src inhibitor or afocal adhesion kinase (FAK) modulator. Although not wishing to be boundby theory, it is believed that the administration of kinase inhibitorsprevents apoptosis of cochlear hair cells, thereby preventing hearingloss. In one embodiment, administration of a compound of the inventionis administered to a subject suffering from hearing loss in order toprevent further hearing loss. In another embodiment, administration of acompound of the invention is administered to a subject suffering fromhearing loss in order to restore lost hearing. In particular, followingnoise exposure, the tight cell junctures between the cochlear haircells, as well as the cell-extracellular matrix interaction, are tornand stressed. The stressing of these tight cell junctures initiatesapoptosis in the cells through a complex signaling pathway in whichtyrosine kinases act as molecular switches, interacting with focaladhesion kinase to transduce signals of cell-matrix disruptions to thenucleus. It is believed that the administration of kinase inhibitorsprevents the initiation of apoptosis in this cascade.

The identification of apoptosis in the noise-exposed cochlea hasgenerated a number of new possibilities for the prevention ofnoise-induced hearing loss (NIHL) (Hu, et al.; 2000, Acta. Otolaryngol.,120, 19-24). For example, the ear can be protected from NIHL byadministration of antioxidant drugs to the round window of the ear(Hight, et al.; 2003, Hear. Res., 179, 21-32; Hu, et al.; Hear. Res.113, 198-206). Specifically, NIHL has been reduced by the administrationof FDA-approved antioxidant compounds (N-L-acetylcysteine (L-NAC) andsalicylate) in the chinchilla (Kopke, et al.; 2000, Hear. Res., 149,138-146). Moreover, Harris et al. have recently described prevention ofNIHL with Src-PTK inhibitors (Harris, et al.; 2005, Hear. Res., 208,14-25). Thus, it is hypothesized that the administration of a compoundof the instant invention which modulates the activity of kinases, isuseful for treating hearing loss.

Changes in cell attachment or cell stress can activate a variety ofsignals through the activation of integrins and through thephosphorylation of PTKs, including the Src family of tyrosine kinases.Src interactions have been linked to signaling pathways that modify thecytoskeleton and activate a variety of protein kinase cascades thatregulate cell survival and gene transcription (reviewed in Giancotti andRuoslahti; 1999, Science, 285, 1028-1032). In fact, recent results haveindicated that outer hair cells (OHC), which had detached at the cellbase following an intense noise exposure, underwent apoptotic celldeath. Specifically, the Src PTK signaling cascade is thought to beinvolved in both metabolic- and mechanically-induced initiation ofapoptosis in sensory cells of the cochlea. In a recent study, Srcinhibitors provided protection from a 4 hour, 4 kHz octave band noise at106 dB, indicating that Src-PTKs might be activated in outer hair cellsfollowing noise exposure (Harris, et al.; 2005, Hear. Res., 208, 14-25).Thus, compounds of the instant invention that modulate the activity ofSrc, are useful in treating hearing loss.

The present invention relates to a method for protecting against ortreating osteoporosis in a subject. Another aspect of the inventionincludes use of a compound of the invention in the manufacture of amedicament to protect against or treat osteoporosis. This methodinvolves administering an effective amount of a compound of theinvention to the subject to protect against or to treat osteoporosis. Inorder to protect against osteoporosis, the compound may be administeredprior to the development of osteoporosis. Alternatively, the compoundmay be used to treat osteoporosis in a subject. In this embodiment, thecompound is administered to the subject subsequent to the initiation ofosteoporosis to reduce the level of osteoporosis.

A compound of the invention can be, e.g. a non-ATP competitiveinhibitor. The compound of the invention can modulate a kinase signalingcascade, depending upon the particular side chains and scaffoldmodifications selected. The compound of the invention can be a kinaseinhibitor. For example, the compound can be a protein tyrosine kinase(PTK) inhibitor. The proline-rich tyrosine kinase (PYK2; also known ascell adhesion kinase (3, related adhesion focal tyrosine kinase, orcalcium-dependent tyrosine kinase) and focal adhesion kinase (FAK) aremembers of a distinct family of non receptor protein-tyrosine kinasesthat are regulated by a variety of extracellular stimuli (Avraham, etal.; 2000, Cell Signal., 12, 123-133; Schlaepfer, et al.; 1999, Prog.Biophys. Mol. Biol., 71, 435-478). The compound of the invention can bea Src inhibitor. It has been shown that Src deficiency is associatedwith osteoporosis in mice, because of loss of osteoclast function(Soriano, et al.; 1991, Cell, 64, 693-702). Alternatively, the compoundof the invention can modulate the expression of interleukin-1 receptorassociated kinase M (IRAK-M). Mice that lack IRAK-M develop severeosteoporosis, which is associated with the accelerated differentiationof osteoclasts, an increase in the half-life of osteoclasts, and theiractivation (Hongmei, et al.; 2005, J. Exp. Med., 201, 1169-1177).

Multinucleated osteoclasts originate from the fusion of mononuclearphagocytes and play a major role in bone development and remodeling viathe resorption of bone. Osteoclasts are multinucleated, terminallydifferentiated cells that degrade mineralized matrix. In normal bonetissue, there is a balance between bone formation by osteoblasts andbone resorption by osteoclasts. When the balance of this dynamic andhighly regulated process is disrupted, bone resorption can exceed boneformation resulting in quantitative bone loss. Because osteoclasts areessential for the development and remodeling of bone, increases in theirnumber and/or activity lead to diseases that are associated withgeneralized bone loss (e.g., osteoporosis) and others with localizedbone loss (e.g., rheumatoid arthritis, periodontal disease).

Osteoclasts and osteoblasts both command a multitude of cellularsignaling pathways involving protein kinases. Osteoclast activation isinitiated by adhesion to bone, cytoskeletal rearrangement, formation ofthe sealing zone, and formation of the polarized ruffled membrane. It isbelieved that protein-tyrosine kinase 2 (PYK2) participates in thetransfer of signals from the cell surface to the cytoskeleton, as it istyrosine phosphorylated and activated by adhesion-initiated signaling inosteoclasts (Duong, et al.; 1998, J. Clin. Invest., 102, 881-892).Recent evidence has indicated that the reduction of PYK2 protein levelsresults in the inhibition of osteoclast formation and bone resorption invitro (Duong, et al.; 2001, J. Bio. Chem., 276, 7484-7492). Therefore,the inhibition of PYK2 or other protein tyrosine kinases might reducethe level of osteoporosis by decreasing osteoclast formation and boneresorption. Thus, without wishing to be bound by theory, it ishypothesized that the administration of a compound of the instantinvention will modulate kinase (e.g. PTK) activity and therefore resultin the inhibition of osteoclast formation and/or bone resorption,thereby treating osteoporosis.

Src tyrosine kinase stands out as a promising therapeutic target forbone disease as validated by Src knockout mouse studies and in vitrocellular experiments, suggesting a regulatory role for Src in bothosteoclasts (positive) and osteoblasts (negative). In osteoclasts, Srcplays key roles in motility, polarization, survival, activation (ruffledborder formation) and adhesion, by mediating various signal transductionpathways, especially in cytokine and integrin signaling (Parang and Sun;2005, Expert Opin. Ther. Patents, 15, 1183-1207). Moreover, targeteddisruption of the src gene in mice induces osteopetrosis, a disordercharacterized by decreased bone resorption, without showing any obviousmorphological or functional abnormalities in other tissues or cells(Soriano, et al.; 1991, Cell, 64, 693-702). The osteopetrotic phenotypeof src^(−/−) mice is cell-autonomous and results from defects in matureosteoclasts, which normally express high levels of Src protein (Home, etal.; 1991, Cell, 119, 1003-1013). By limiting the effectiveness of Srctyrosine kinase, which triggers osteoclast activity and inhibitsosteoblasts, Src inhibitors are thought to lessen bone break down andencourage bone formation. Because osteoclasts normally express highlevels of Src, inhibition of Src kinase activity might be useful in thetreatment of osteoporosis (Missbach, et al.; 1999, Bone, 24, 437-449).Thus, the PTK inhibitors of the instant invention that modulate theactivity of Src, are useful in treating osteoporosis.

As described herein, a compound of the invention may be used to protectagainst or prevent obesity in a subject. Another aspect of the inventionincludes use of a compound of the invention in the manufacture of amedicament to protect against or treat obesity. In order to protectagainst obesity, the compound may be administered prior to thedevelopment of obesity in a subject. Alternatively, the compound may beused to treat obesity in a subject. A compound of the instant inventionmay be involved in modulating a kinase signaling cascade, e.g., a kinaseinhibitor, a non-ATP competitive inhibitor, a tyrosine kinase inhibitor,a protein tyrosine phosphatase inhibitor, or a protein-tyrosinephosphatase 1B inhibitor.

Obesity is associated with diabetes and increased insulin resistance ininsulin responsive tissues, such as skeletal muscle, liver, and whiteadipose tissue (Klaman, et al.; 2000, Mol. Cell. Biol., 20, 5479-5489).Insulin plays a critical role in the regulation of glucose homeostasis,lipid metabolism, and energy balance. Insulin signaling is initiated bybinding of insulin to the insulin receptor (IR), a receptor tyrosinekinase. Insulin binding evokes a cascade of phosphorylation events,beginning with the autophosphorylation of the IR on multiple tyrosylresidues. Autophosphorylation enhances IR kinase activity and triggersdownstream signaling events. The stimulatory effects of protein tyrosinekinases and the inhibitory effects of protein tyrosine phosphataseslargely define the action of insulin. Appropriate insulin signalingminimizes large fluctuations in blood glucose concentrations and ensuresadequate delivery of glucose to cells. Since insulin stimulation leadsto multiple tyrosyl phosphorylation events, enhanced activity of one ormore protein-tyrosine phosphatases (PTPs) could lead to insulinresistance, which may lead to obesity. Indeed, increased PTP activityhas been reported in several insulin-resistant states, including obesity(Ahmad, et al.; 1997, Metabolism, 46, 1140-1145). Thus, without wishingto be bound by theory, the administration of a compound of the instantinvention modulates kinase (e.g., PTP) activity, thereby treatingobesity in a subject.

Insulin signaling begins with the activation of the IR via tyrosinephosphorylation and culminates in the uptake of glucose into cells bythe glucose transporter, GLUT4 (Saltiel and Kahn; 2001, Nature, 414,799-806). The activated IR must then be deactivated and returned to abasal state, a process that is believed to involve protein-tyrosinephosphatase-1B (PTP-1B) (Ahmad, et al; 1997, J. Biol. Chem., 270,20503-20508). Disruption of the gene that codes for PTP-1B in miceresults in sensitivity to insulin and increased resistance todiet-induced obesity (Elchebly, et al.; 1999, Science, 283, 1544-1548;Klaman, et al.; 2000, Mol. Cell. Biol., 20, 5479-5489). The decreasedadiposity in PTP-1B deficient mice was due to a marked reduction in fatcell mass without a decrease in adipocyte number (Klaman, et al.; 2000,Mol. Cell. Biol., 20, 5479-5489). Moreover, leanness in PTP-1B-deficientmice was accompanied by increased basal metabolic rate and total energyexpenditure, without marked alteration of uncoupling protein mRNAexpression. The disruption of the PTP-1B gene demonstrated that alteringthe activity of PTP-1B can modulate insulin signaling anddietary-induced obesity in vivo. Thus, without wishing to be bound bytheory, the administration of a compound of the instant invention thatmodulates insulin signaling (e.g., PTP-1B activity), is useful intreating obesity in a subject.

As described herein, a compound of the invention may be used to protectagainst or prevent diabetes in a subject. Another aspect of theinvention includes use of a compound of the invention in the manufactureof a medicament to protect against or prevent diabetes. In order toprotect against diabetes, the compound may be administered prior to thedevelopment of diabetes in a subject. Alternatively, the compound may beused to treat diabetes in a subject. The compound of the instantinvention may be involved in modulating a kinase signaling cascade, e.g.a kinase inhibitor, a non-ATP competitive inhibitor, a tyrosine kinaseinhibitor, a phosphatase and tension homologue on chromosome 10 (PTEN)inhibitor, or a sequence homology 2-containing inositol 5′-phosphatase 2(SHIP2) inhibitor.

Type 2 diabetes mellitus (T2DM) is a disorder of dysregulated energymetabolism. Energy metabolism is largely controlled by the hormoneinsulin, a potent anabolic agent that promotes the synthesis and storageof proteins, carbohydrates and lipids, and inhibits their breakdown andrelease back into the circulation. Insulin action is initiated bybinding to its tyrosine kinase receptor, which results inautophosphorylation and increased catalytic activity of the kinase(Patti, et al.; 1998, J Basic Clin. Physiol. Pharmacol. 9, 89-109).Tyrosine phosphorylation causes insulin receptor substrate (IRS)proteins to interact with the p85 regulatory subunit ofphosphatidylinositol 3-kinase (PI3K), leading to the activation of theenzyme and its targeting to a specific subcellular location, dependingon the cell type. The enzyme generates the lipid productphosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃), whichregulates the localization and activity of numerous proteins (Kido, etal.; 2001, J. Clin. Endocrinol. Metab., 86, 972-979). PI3K has anessential role in insulin-stimulated glucose uptake and storage,inhibition of lipolysis and regulation of hepatic gene expression(Saltiel, et al.; 2001, Nature, 414, 799-806). Overexpression ofdominant-interfering forms of PI3K can block glucose uptake andtranslocation of glutamate transporter four, GLUT4, to the plasmamembrane (Quon, et al.; 1995, Mol. Cell. Biol., 15, 5403-5411). Thus,the administration of a compound of the instant invention that modulateskinase (e.g. PI3K) activity, and therefore results in increased glucoseuptake, is useful in treating diabetes.

PTEN is a major regulator of PI3K signaling in may cell types, andfunctions as a tumor suppressor due to antagonism of the anti-apoptotic,proliferative and hypertrophic activities of the PI3K pathway(Goberdhan, et al.; 2003, Hum. Mol. Genet., 12, R239-R248; Leslie, etal.; 2004, J. Biochem., 382, 1-11). Although not wishing to be bound bytheory, it is believed that PTEN attenuates the PI3K pathway bydephosphorylation of the PtdIns(3,4,5)P3 molecule, degrading thisimportant lipid second messenger to PtdIns(4,5)P2. In a recent study,reduction of endogenous PTEN protein by 50% using small interfering RNA(siRNA) enhanced insulin-dependent increases in PtdIns(3,4,5)P3 levels,and glucose uptake (Tang, et al.; 2005, J. Biol. Chem., 280,22523-22529). Thus, without wishing to be bound by theory, it ishypothesized that the administration of a compound of the instantinvention that modulates PTEN activity, and therefore results inincreased glucose uptake, is useful for treating diabetes.

PtdIns(3,4,5)P3 levels are also controlled by the family of SRC homology2 (SH2)-containing inositol 5′-phosphatase (SHIP) proteins, SHIP1 andSHIP2 (Lazar and Saltiel; 2006, Nature Reviews, 5, 333-342). SHIP2,expressed in skeletal muscle, among other insulin-sensitive tissues,catalyzes the conversion of PtdIns(3,4,5)P3 into PtdIns(3,4)P2 (Pesesse,et al.; 1997; Biochem Biophys. Res. Commun., 239, 697-700; Backers, etal.; 2003, Adv. Enzyme Regul., 43, 15-28; Chi, et al.; 2004, J Biol.Chem., 279, 44987-44995; Sleeman, et al.; 2005, Nature Med., 11,199-205). Overexpression of SHIP2 markedly reduced insulin-stimulatedPtdIns(3,4,5)P3 levels, consistent with the proposed capacity of SHIP2to attenuate the activation of downstream effectors of PI3K (Ishihara,et al.; 1999, Biochem. Biophys. Res. Commun., 260, 265-272). Thus,without wishing to be bound by theory, it is hypothesized that theadministration of a compound of the instant invention which modulatesSHIP2 activity, and therefore results in increased glucose uptake, isuseful for treating diabetes.

As described herein, a compound of the invention may be used to protectagainst or prevent eye disease in a subject. Another aspect of theinvention includes use of a compound of the invention in the manufactureof a medicament to protect against or prevent eye disease. In order toprotect against eye disease, the compound may be administered prior tothe development of eye disease in a subject. Alternatively, the compoundmay be used to treat eye disease in a subject, e.g. maculardegeneration, retinopathy, and macular edema. The compound of theinstant invention may be involved in modulating a kinase cascade, e.g. akinase inhibitor, a non-ATP competitive inhibitor, a tyrosine kinaseinhibitor, e.g. a vascular endothelial growth factor (VEGF) receptortyrosine kinase inhibitor.

Vision-threatening neovascularization of the physiologically avascularcornea can occur. The proliferative retinopathies, principally diabeticretinopathy and age-related macular degeneration, are characterized byincreased vascular permeability, leading to retinal edema and subretinalfluid accumulation, and the proliferation of new vessels that are proneto hemorrhage. Angiogenesis, the formation of new blood vessels frompreexisting capillaries, is an integral part of both normal developmentand numerous pathological processes. VEGF, a central mediator of thecomplex cascade of angiogenesis and a potent permeability factor, is anattractive target for novel therapeutics. VEGF is the ligand for twomembrane-bound tyrosine kinase receptors, VEGFR-1 and VEGFR-2. Ligandbinding triggers VEGFR dimerization and transphosphorylation withsubsequent activation of an intracellular tyrosine kinase domain. Theensuing intracellular signaling axis results in vascular endothelialcell proliferation, migration, and survival. Thus, without wishing to bebound by theory, it is hypothesized that the administration of acompound of the instant invention which modulates kinase activity, e.g.tyrosine kinase activity, and results in the inhibition of angiogenesisand/or neovascularization, is useful for treating an eye disease, e.g.macular degeneration, retinopathy and/or macular edema.

Macular degeneration is characterized by VEGF-mediated retinal leakage(an increase in vascular permeability) and by the abnormal growth ofsmall blood vessels in the back of the eye (angiogenesis). VEGF has beenidentified in neovascular membranes in both diabetic retinopathy andage-related macular degeneration, and intraocular levels of the factorcorrelate with the severity of neovascularization in diabeticretinopathy (Kvanta, et al.; 1996, Invest. Ophthal. Vis. Sci., 37,1929-1934.; Aiello, et al.; 1994, N. Engl. J Med., 331, 1480-1487).Therapeutic antagonism of VEGF in these models results in significantinhibition of both retinal and choroidal neovascularization, as well asa reduction in vascular permeability (Aiello, et al.; 1995, Proc. Natl.Acad. Sci. USA., 92, 10457-10461; Krzystolik, et al.; 2002, Arch.Ophthal., 120, 338-346; Qaum, et al.; 2001, Invest. Ophthal. Vis. Sci.,42, 2408-2413). Thus, without wishing to be bound by theory, it ishypothesized that the administration of a compound of the instantinvention which modulates VEGF activity, and results in the inhibitionof angiogenesis and/or neovascularization, is useful for treating an eyedisease, e.g. macular degeneration, retinopathy and/or macular edema.

The compounds of the invention are used in methods of treating,preventing, or ameliorating a stroke in a subject who is at risk ofsuffering a stroke, is suffering from a stroke or has suffered a stroke.The compounds of the invention are useful in methods of treatingpatients who are undergoing post-stroke rehabilitation. Another aspectof the invention includes use of a compound of the invention in themanufacture of a medicament to treat, prevent, or ameliorating stroke.

A stroke, also known as a cerebrovascular accident (CVA), is an acuteneurological injury whereby the blood supply to a part of the brain isinterrupted due to either blockage of an artery or rupture of a bloodvessel. The part of the brain in which blood supply is interrupted nolonger receives oxygen and/or nutrients carried by the blood. The braincells become damaged or necrotic, thereby impairing function in or fromthat part of the brain. Brain tissue ceases to function if deprived ofoxygen for more than 60 to 90 seconds and after a few minutes willsuffer irreversible injury possibly leading to a death of the tissue,i.e., infarction.

Strokes are classified into two major types: ischemic, i.e., blockage ofa blood vessel supplying the brain, and hemorrhagic, i.e., bleeding intoor around the brain. The majority of all strokes are ischemic strokes.Ischemic stroke is commonly divided into thrombotic stroke, embolicstroke, systemic hypoperfusion (Watershed stroke), or venous thrombosis.In thrombotic stroke, a thrombus-forming process develops in theaffected artery, the thrombus, i.e., blood clot, gradually narrows thelumen of the artery, thereby impeding blood flow to distal tissue. Theseclots usually form around atherosclerotic plaques. There are two typesof thrombotic strokes, which are categorized based on the type of vesselon which the thrombus is formed. Large vessel thrombotic stroke involvesthe common and internal carotids, vertebral, and the Circle of Willis.Small vessel thrombotic stroke involves the intracerebral arteries,branches of the Circle of Willis, middle cerebral artery stem, andarteries arising from the distal vertebral and basilar artery.

A thrombus, even if non-occluding, can lead to an embolic stroke if thethrombus breaks off, at which point it becomes an embolus. An embolusrefers to a traveling particle or debris in the arterial bloodstreamoriginating from elsewhere. Embolic stroke refers to the blockage ofarterial access to a part of the brain by an embolus. An embolus isfrequently a blood clot, but it can also be a plaque that has broken offfrom an atherosclerotic blood vessel or a number of other substancesincluding fat, air, and even cancerous cells. Because an embolus arisesfrom elsewhere, local therapy only solves the problem temporarily. Thus,the source of the embolus must be identified. There are four categoriesof embolic stroke: those with a known cardiac source; those with apotential cardiac or aortic source (from trans-thoracic ortrans-esophageal echocardiogram); those with an arterial source; andthose with unknown source.

Systemic hypoperfusion is the reduction of blood flow to all parts ofthe body. It is most commonly due to cardiac pump failure from cardiacarrest or arrhythmias, or from reduced cardiac output as a result ofmyocardial infarction, pulmonary embolism, pericardial effusion, orbleeding. Hypoxemia (i.e., low blood oxygen content) may precipitate thehypoperfusion. Because the reduction in blood flow is global, all partsof the brain may be affected, especially the “watershed” areas which areborder zone regions supplied by the major cerebral arteries. Blood flowto these area has not necessary stopped, but instead may have lessenedto the point where brain damage occurs.

Veins in the brain function to drain the blood back to the body. Whenveins are occluded due to thrombosis, the draining of blood is blockedand the blood backs up, causing cerebral edema. This cerebral edema canresult in both ischemic and hemorrhagic strokes. This commonly occurs inthe rare disease sinus vein thrombosis.

Stroke is diagnosed in a subject or patient using one or more of avariety of techniques known in the art, such as, for example,neurological examination, blood tests, CT scans (without contrastenhancements), MRI scans, Doppler ultrasound, and arteriography (i.e.,roentgenography of arteries after injection of radiopacque material intothe blood stream). If a stroke is confirmed on imaging, various otherstudies are performed to determine whether there is a peripheral sourceof emboli. These studies include, e.g., an ultrasound/doppler study ofthe carotid arteries (to detect carotid stenosis); an electrocardiogram(ECG) and echocardiogram (to identify arrhythmias and resultant clots inthe heart which may spread to the brain vessels through thebloodstream); a Holter monitor study to identify intermittentarrhythmias and an angiogram of the cerebral vasculature (if a bleed isthought to have originated from an aneurysm or arteriovenousmalformation).

Compounds useful in these methods of treating, preventing orameliorating stroke or a symptom associated with stroke are compoundsthat modulate kinase signaling cascade preceding, during or after astroke. In some embodiments, the compound is a kinase inhibitor. Forexample, the compound is a tyrosine kinase inhibitor. In an embodiment,the tyrosine kinase inhibitor is an Src inhibitor. Preferably, thecompound used in the methods of treating, preventing or amelioratingstroke or a symptom associated with stroke described herein is anallosteric inhibitor of kinase signaling cascade preceding, during orafter a stroke. Preferably, the compound used in the methods oftreating, preventing or ameliorating stroke or a symptom associated withstroke described herein is a non-ATP competitive inhibitor of kinasesignaling cascade preceding, during or after a stroke.

Inhibition of Src activity has been shown to provide cerebral protectionduring stroke. (See Paul et al., Nature Medicine, vol. 7(2):222-227(2001), which is hereby incorporated by reference in its entirety).Vascular endothelia growth factor (VEGF), which is produced in responseto the ischemic injury, has been shown to promote vascular permeability.Studies have shown that the Src kinase regulates VEGF-mediated VP in thebrain following stroke, and administration of an Src inhibitor beforeand after stroke reduced edema, improved cerebral perfusion anddecreased infarct volume after injury occurred. (Paul et al., 2001).Thus, Src inhibition may be useful in the prevention, treatment oramelioration of secondary damage following a stroke.

The compounds of the invention prevent, treat or ameliorate stroke or asymptom associated with stroke. Another aspect of the invention includesuse of a compound of the invention in the manufacture of a medicament toprevent, treat, or ameliorate stroke or a symptom associated withstroke. Symptoms of a stroke include sudden numbness or weakness,especially on one side of the body; sudden confusion or trouble speakingor understanding speech; sudden trouble seeing in one or both eyes;sudden trouble with walking, dizziness, or loss of balance orcoordination; or sudden severe headache with no known cause.

Generally there are three treatment stages for stroke: prevention,therapy immediately after the stroke, and post-stroke rehabilitation.Therapies to prevent a first or recurrent stroke are based on treatingthe underlying risk factors for stroke, such as, e.g., hypertension,high cholesterol, atrial fibrillation, and diabetes. Acute stroketherapies try to stop a stroke while it is happening by quicklydissolving the blood clot causing an ischemic stroke or by stopping thebleeding of a hemorrhagic stroke. Post-stroke rehabilitation helpsindividuals overcome disabilities that result from stroke damage.Medication or drug therapy is the most common treatment for stroke. Themost popular classes of drugs used to prevent or treat stroke areanti-thrombotics (e.g., anti-platelet agents and anticoagulants) andthrombolytics. The compounds are administered to a patient who is atrisk of suffering a stroke, is suffering from a stroke or has suffered astroke at a time before, during, after, or any combination thereof, theoccurrence of a stroke. The compounds of the invention are administeredalone, in pharmaceutical compositions, or in combination with any of avariety of known treatments, such as, for example, an anti-plateletmedication (e.g., aspirin, clopidogrel, dipyridamole), an anti-coagulant(e.g., warfarin), or a thrombolytic medication (e.g., tissue plasminogenactivator (t-PA), reteplase, Urokinase, streptokinase, tenectaplase,lanoteplase, or anistreplase.

The compounds of the invention are used in methods of treating,preventing, ameliorating atherosclerosis or a symptom thereof in asubject who is at risk for or suffering from atherosclerosis. Anotheraspect of the invention includes use of a compound of the invention inthe manufacture of a medicament to treat, prevent, or ameliorateatherosclerosis.

Atherosclerosis is a disease affecting the arterial blood vessel and iscommonly referred to as a “hardening” of the arteries. It is caused bythe formation of multiple plaques within the arteries. Atheroscleroticplaques, though compensated for by artery enlargement, eventually leadto plaque ruptures and stenosis (i.e., narrowing) of the artery, which,in turn, leads to an insufficient blood supply to the organ it feeds.Alternatively, if the compensating artery enlargement process isexcessive, a net aneurysm results. These complications are chronic,slowly progressing and cumulative. Most commonly, soft plaque suddenlyruptures, causing the formation of a blood clot (i.e., thrombus) thatrapidly slows or stops blood flow, which, in turn, leads to death of thetissues fed by the artery. This catastrophic event is called aninfarction. For example, coronary thrombosis of a coronary artery causesa myocardial infarction, commonly known as a heart attack. A myocardialinfarction occurs when an atherosclerotic plaque slowly builds up in theinner lining of a coronary artery and then suddenly ruptures, totallyoccluding the artery and preventing blood flow downstream.

Atherosclerosis and acute myocardial infarction are diagnosed in apatient using any of a variety of clinical and/or laboratory tests suchas, physical examination, radiologic or ultrasound examination and bloodanalysis. For example, a doctor or clinical can listen to a subject'sarteries to detect an abnormal whooshing sound, called a bruit. A bruitcan be heard with a stethoscope when placed over the affected artery.Alternatively, or in addition, the clinician or physician can checkpulses, e.g., in the leg or foot, for abnormalities such as weakness orabsence. The physician or clinical may perform blood work to check forcholesterol levels or to check the levels of cardiac enzymes, such ascreatine kinase, troponin and lactate dehydrogenase, to detectabnormalities. For example, troponin sub-units I or T, which are veryspecific for the myocardium, rise before permanent injury develops. Apositive troponin in the setting of chest pain may accurately predict ahigh likelihood of a myocardial infarction in the near future. Othertests to diagnose atherosclerosis and/or myocardial infarction include,for example, EKG (electrocardiogram) to measure the rate and regularityof a subject's heartbeat; chest X-ray, measuring ankle/brachial index,which compares the blood pressure in the ankle with the blood pressurein the arm; ultrasound analysis of arteries; CT scan of areas ofinterest; angiography; an exercise stress test, nuclear heart scanning;and magnetic resonance imaging (MRI) and positron emission tomography(PET) scanning of the heart.

Compounds useful in these methods of treating, preventing orameliorating atherosclerosis or a symptom thereof are compounds thatmodulate kinase signaling cascade in a patient at risk for or sufferingfrom atherosclerosis. In some embodiments, the compound is a kinaseinhibitor. For example, the compound is a tyrosine kinase inhibitor. Inan embodiment, the tyrosine kinase inhibitor is an Src inhibitor.Preferably, the compound used in the methods of treating, preventing orameliorating atherosclerosis or a symptom thereof described herein is anallosteric inhibitor of kinase signaling cascade involved inatherosclerosis. Preferably, the compound used in the methods oftreating, preventing or ameliorating atherosclerosis or a symptomassociated with atherosclerosis described herein is a non-ATPcompetitive inhibitor of kinase signaling cascade involved inatherosclerosis.

Cellular signal transduction by Src is believed to play a key role inincreased permeability of vessels, known as vascular permeability (VP).Vascular endothelia growth factor (VEGF), which is produced in responseto the ischemic injury, including, e.g., myocardial infarction, has beenshown to promote vascular permeability. Studies have shown that theinhibition of Src kinase decreases VEGF-mediated VP. (See Parang andSun, Expert Opin. Ther. Patents, vol. 15(9): 1183-1206 (2005), which ishereby incorporated by reference in its entirety). Mice treated with anSrc inhibitor demonstrated reduced tissue damage associated with traumaor injury to blood vessels after myocardial infarction, as compared tountreated mice. (See e.g., U.S. Patent Publication Nos. 20040214836 and20030130209 by Cheresh et al., the contents of which are herebyincorporated by reference in their entirety). Thus, Src inhibition maybe useful in the prevention, treatment or amelioration of secondarydamage following injury due to atherosclerosis, such as, for example,myocardial infarction.

The compounds of the invention prevent, treat or ameliorate stroke or asymptom associated with atherosclerosis. Another aspect of the inventionincludes use of a compound of the invention in the manufacture of amedicament to prevent, treat, or ameliorate stroke or a symptomassociated with atherosclerosis. Atherosclerosis generally does notproduce symptoms until it severely narrows the artery and restrictsblood flow, or until it causes a sudden obstruction. Symptoms depend onwhere the plaques and narrowing develop, e.g., in the heart, brain,other vital organs and legs or almost anywhere in the body. The initialsymptoms of atherosclerosis may be pain or cramps when the body requiresmore oxygen, for example during exercise, when a person may feel chestpain (angina) because of lack of oxygen to the heart or leg crampsbecause of lack of oxygen to the legs. Narrowing of the arteriessupplying blood to the brain may cause dizziness or transient ischemicattacks (TIA's) where the symptoms and signs of a stroke last less than24 hours. Typically, these symptoms develop gradually.

Symptoms of myocardial infarction are characterized by varying degreesof chest pain, discomfort, sweating, weakness, nausea, vomiting, andarrhythmias, sometimes causing loss of consciousness. Chest pain is themost common symptom of acute myocardial infarction and is oftendescribed as a tightness, pressure, or squeezing sensation. Pain mayradiate to the jaw, neck, arms, back, and epigastrium, most often to theleft arm or neck. Chest pain is more likely caused by myocardialinfarction when it lasts for more than 30 minutes. Patients sufferingfrom a myocardial infarction may exhibit shortness of breath (dyspnea)especially if the decrease in myocardial contractility due to theinfarct is sufficient to cause left ventricular failure with pulmonarycongestion or even pulmonary edema.

The compounds of the invention are administered alone, in pharmaceuticalcompositions, or in combination with any of a variety of knowntreatments for atherosclerosis, such as, for example,cholesterol-lowering drugs (e.g., statins), anti-platelet medications,or anti-coagulants.

The compounds of the invention are used in methods of treating,preventing, ameliorating neuropathic pain, such as chronic neuropathicpain, or a symptom thereof in a subject who is at risk of sufferingfrom, is suffering from, or has suffered neuropathic pain. Anotheraspect of the invention includes use of a compound of the invention inthe manufacture of a medicament to treat, prevent or ameliorateneuropathic pain.

Neuropathic pain, also known as neuralgia, is qualitatively differentfrom ordinary nociceptive pain. Neuropathic pain usually presents as asteady burning and/or “pins and needles” and/or “electric shock”sensations. The difference between nociceptive pain and neuropathic painis due to the fact that “ordinary”, nociceptive pain stimulates onlypain nerves, while a neuropathy often results in the stimulation of bothpain and non-pain sensory nerves (e.g., nerves that respond to touch,warmth, cool) in the same area, thereby producing signals that thespinal cord and brain do not normally expect to receive.

Neuropathic pain is a complex, chronic pain state that usually isaccompanied by tissue injury. With neuropathic pain, the nerve fibersthemselves may be damaged, dysfunctional or injured. These damaged nervefibers send incorrect signals to other pain centers. The impact of nervefiber injury includes a change in nerve function both at the site ofinjury and areas around the injury.

Neuropathic pain is diagnosed in a subject or patient using one or moreof a variety of laboratory and/or clinical techniques known in the art,such as, for example, physical examination.

Compounds useful in these methods of treating, preventing orameliorating neuropathic pain, such as chronic neuropathic pain, or asymptom associated with neuropathic pain are compounds that modulatekinase signaling cascade involved in neuropathic pain. In someembodiments, the compound is a kinase inhibitor. For example, thecompound is a tyrosine kinase inhibitor. In an embodiment, the tyrosinekinase inhibitor is an Src inhibitor. Preferably, the compound used inthe methods of treating, preventing or ameliorating neuropathic pain ora symptom thereof is an allosteric inhibitor of kinase signaling cascadeinvolved in neuropathic pain. Preferably, the compound used in themethods of treating, preventing or ameliorating neuropathic pain or asymptom thereof is a non-ATP competitive inhibitor of kinase signalingcascade involved in neuropathic pain.

c-Src has been shown to regulate the activity of N-methyl-D-aspartate(NMDA) receptors. (See Yu et al., Proc. Natl. Acad. Sci. USA, vol.96:7697-7704 (1999), which is hereby incorporated by reference in itsentirety). Studies have shown that PP2, a low molecular weight Srckinase inhibitor, decreases phosphorylation of the NMDA receptor NM2subunit. (See Guo et al., J. Neuro., vol. 22:6208-6217 (2002), which ishereby incorporated by reference in its entirety). Thus, Src inhibition,which in turn, inhibits the activity NMDA receptors, may be useful inthe prevention, treatment or amelioration of neuropathic pain, such aschronic neuropathic pain.

The compounds of the invention prevent, treat or ameliorate neuropathicpain, such as chronic neuropathic pain, or a symptom associated withneuropathic pain. Symptoms of neuropathic pain include shooting andburning pain, tingling and numbness.

The compounds of the invention are administered alone, in pharmaceuticalcompositions, or in combination with any of a variety of knowntreatments, such as, for example, analgesics, opioids, tricyclicantidepressants, anticonvulsants and serotonin norepinephrine reuptakeinhibitors.

The compounds of the invention are used in methods of treating,preventing, ameliorating hepatitis B or a symptom thereof in a subjectwho is at risk for or suffering from hepatitis B. Another aspect of theinvention includes use of a compound of the invention in the manufactureof a medicament to treat, prevent, or ameliorate hepatitis B.

The hepatitis B virus, a member of the Hepadnavirus family, consists ofa proteinaceous core particle containing the viral genome in the form ofdouble stranded DNA with single-stranded regions and an outerlipid-based envelope with embedded proteins. The envelope proteins areinvolved in viral binding and release into susceptible cells. The innercapsid relocates the DNA genome to the cell's nucleus where viral mRNAsare transcribed. Three subgenomic transcripts encoding the envelopeproteins are made, along with a transcript encoding the X protein. Afourth pre-genomic RNA is transcribed, which is exported to the cytosoland translates the viral polymerase and core proteins. Polymerase andpre-genomic RNA are encapsidated in assembling core particles, wherereverse transcription of the pre-genomic RNA to genomic DNA occurs bythe polymerase protein. The mature core particle then exits the cell vianormal secretory pathways, acquiring an envelope along the way.

Hepatitis B is one of a few known non-retroviral viruses that employreverse transcription as part of the replication process. Other viruseswhich use reverse transcription include, e.g., HTLV or HIV.

During HBV infection, the host immune response is responsible for bothhepatocellular damage and viral clearance. While the innate immuneresponse does not play a significant role in these processes, theadaptive immune response, particularly virus-specific cytotoxic Tlymphocytes (CTLs), contributes to nearly all of the liver injuryassociated with HBV infection. By killing infected cells and byproducing antiviral cytokines capable of purging HBV from viablehepatocytes, CTLs also eliminate the virus. Although liver damage isinitiated and mediated by the CTLs, antigen-nonspecific inflammatorycells can worsen CTL-induced immunopathology and platelets mayfacilitate the accumulation of CTLs into the liver.

Hepatitis B is diagnosed in a patient using any of a variety of clinicaland/or laboratory tests such as, physical examination, and blood orserum analysis. For example, blood or serum is assayed for the presenceof viral antigens and/or antibodies produced by the host. In a commontest for Hepatitis B, detection of hepatitis B surface antigen (HBsAg)is used to screen for the presence of infection. It is the firstdetectable viral antigen to appear during infection with this virus;however, early in an infection, this antigen may not be present and itmay be undetectable later in the infection as it is being cleared by thehost. During this ‘window’ in which the host remains infected but issuccessfully clearing the virus, IgM antibodies to the hepatitis B coreantigen (anti-HBc IGM) may be the only serologic evidence of disease.

Shortly after the appearance of the HBsAg, another antigen named as thehepatitis B e antigen (HBeAg) will appear. Traditionally, the presenceof HBeAg in a host's serum is associated with much higher rates of viralreplication; however, some variants of the hepatitis B virus do notproduce the “e” antigen at all. During the natural course of aninfection, the HBeAg may be cleared, and antibodies to the “e” antigen(anti-HBe) will arise immediately afterward. This conversion is usuallyassociated with a dramatic decline in viral replication. If the host isable to clear the infection, eventually the HBsAg will becomeundetectable and will be followed by antibodies to the hepatitis Bsurface antigen (anti-HBs). A person negative for HBsAg but positive foranti-HBs has either cleared an infection or has been vaccinatedpreviously. A number of people who are positive for HBsAg may have verylittle viral multiplication, and hence may be at little risk oflong-term complications or of transmitting infection to others.

Compounds useful in these methods of treating, preventing orameliorating hepatitis B or a symptom thereof are compounds thatmodulate kinase signaling cascade in a patient at risk for or sufferingfrom hepatitis B. In some embodiments, the compound is a kinaseinhibitor. For example, the compound is a tyrosine kinase inhibitor. Inan embodiment, the tyrosine kinase inhibitor is an Src inhibitor.Preferably, the compound used in the methods of treating, preventing orameliorating hepatitis B or a symptom thereof described herein is anallosteric inhibitor of kinase signaling cascade involved in hepatitisB. Preferably, the compound used in the methods of treating, preventingor ameliorating hepatitis B or a symptom associated with hepatitis Bdescribed herein is a non-ATP competitive inhibitor of kinase signalingcascade involved in hepatitis B.

Src plays a role in the replication of the hepatitis B virus. Thevirally encoded transcription factor HBx activates Src in a step that isrequired from propagation of the HBV virus. (See, e.g., Klein et al.,EMBO J., vol. 18:5019-5027 (1999); Klein et al., Mol. Cell. Biol., vol.17:6427-6436 (1997), each of which is hereby incorporated by referencein its entirety). Thus, Src inhibition, which in turn, inhibitsSrc-mediated propagation of the HBV virus, may be useful in theprevention, treatment or amelioration of hepatitis B or a symptomthereof.

The compounds of the invention prevent, treat or ameliorate hepatitis Bor a symptom associated with hepatitis B. Symptoms of hepatitis Btypically develop within 30-180 days of exposure to the virus. However,up to half of all people infected with the hepatitis B virus have nosymptoms. The symptoms of hepatitis B are often compared to flu, andinclude, e.g., appetite loss; fatigue; nausea and vomiting, itching allover the body; pain over the liver (e.g., on the right side of theabdomen, under the lower rib cage), jaundice, and changes in excretoryfunctions.

The compounds of the invention are administered alone, in pharmaceuticalcompositions, or in combination with any of a variety of knowntreatments for hepatitis B, such as, for example, interferon alpha,lamivudine (Epivir-HBV) and baraclude (entecavir).

As described herein, the compounds of the invention may be used toregulate immune system activity in a subject, thereby protecting againstor preventing autoimmune disease, e.g., rheumatoid arthritis, multiplesclerosis, sepsis and lupus as well as transplant rejection and allergicdiseases. Another aspect of the invention includes use of a compound ofthe invention in the manufacture of a medicament to regulate the immunesystem. Alternatively, the compound may be used to treat autoimmunedisease in a subject. For example, the compound may result in reductionin the severity of symptoms or halt impending progression of theautoimmune disease in a subject. The compound of the invention may beinvolved in modulating a kinase signaling cascade, e.g., a kinaseinhibitor, a non-ATP competitive inhibitor, a tyrosine kinase inhibitor,e.g., a Src inhibitor, a p59fyn (Fyn) inhibitor or a p56lck (Lck)inhibitor.

Autoimmune diseases are diseases caused by a breakdown of self-tolerancesuch that the adaptive immune system responds to self antigens andmediates cell and tissue damage. Autoimmune diseases can be organspecific (e.g., thyroiditis or diabetes) or systemic (e.g., systemiclupus erythematosus). T cells modulate the cell-mediated immune responsein the adaptive immune system. Under normal conditions, T cells expressantigen receptors (T cell receptors) that recognize peptide fragments offoreign proteins bound to self major histocompatibility complexmolecules. Among the earliest recognizable events after T cell receptor(TCR) stimulation are the activation of Lck and Fyn, resulting in TCRphosphorylation on tyrosine residues within immunoreceptortyrosine-based activation motifs (Zamoyska, et al.; 2003, Immunol. Rev.,191, 107-118). Tyrosine kinases, such as Lck (which is a member of theSrc family of protein tyrosine kinases) play an essential role in theregulation of cell signaling and cell proliferation by phosphorylatingtyrosine residues of peptides and proteins (Levitzki; 2001, Top. Curr.Chem., 211, 1-15; Longati, et al.; 2001, Curr. Drug Targets, 2, 41-55;Qian, and Weiss; 1997, Curr. Opin. Cell Biol., 9, 205-211). Thus,although not wishing to be bound by theory, it is hypothesized that theadministration of a compound of the instant invention which modulatestyrosine kinase (e.g., Src) activity is useful in the treatment ofautoimmune disease.

The tyrosine kinases lck and fyn are both activated in the TCR pathway;thus, inhibitors of lck and/or fyn have potential utility as autoimmuneagents (Palacios and Weiss; 2004, Oncogene, 23, 7990-8000). Lck and Fynare predominantly expressed by T cells through most of their lifespan.The roles of Lck and Fyn in T cell development, homeostasis andactivation have been demonstrated by animal and cell line studies(Parang and Sun; 2005, Expert Opin. The. Patents, 15, 1183-1207). Lckactivation is involved in autoimmune diseases and transplant rejection(Kamens, et al.; 2001, Curr. Opin. Investig. Drugs, 2, 1213-1219).Results have shown that the lck (-) Jurkat cell lines are unable toproliferate, produce cytokines, and generate increases in intracellularcalcium, inositol phosphate, and tyrosine phosphorylation in response toT cell receptor stimulation (Straus and Weiss; 1992, Cell., 70, 585-593;Yamasaki, et al.; 1996, Mol. Cell. Biol., 16, 7151-7160). Therefore, anagent inhibiting lck would effectively block T cell function, act as animmunosuppressive agent, and have potential utility in autoimmunediseases, such as rheumatoid arthritis, multiple sclerosis, and lupus,as well as in the area of transplant rejection and allergic diseases(Hanke and Pollok; 1995, Inflammation Res., 44, 357-371). Thus, althoughnot wishing to be bound by theory, it is hypothesized that theadministration of a compound of the instant invention which modulatesone or more members of the Src family of protein tyrosine kinases (e.g.,lck and/or fyn) is useful in the treatment of autoimmune disease.

Compounds of the invention include compounds with water solubilizinggroups appended on the compound (Wermuth, C.G., The Practice ofMedicinal Chemistry 2003, p. 617). e.g., SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂,amines,

tetrazole, etc.

Compounds of the invention include compounds of Formula I, and saltsthereof:

where:

T is absent (i.e., the rings are connected by a bond), CR₁₂R₁₃, C(O), O,S, S(O), S(O)₂, NR₁₄, C(R₁₅R₁₆)C(R₁₇R₁₈), CH₂O, or OCH₂;

X_(y) is CZ, CY, N, or N—O;

X_(z) is CZ, CY, N, or N—O;

at least one of X_(y) and X_(z) is CZ;

Y is selected from hydrogen, hydroxyl, halogen, lower (C₁, C₂, C₃, C₄,C₅, or C₆) alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃,C₄, C₅, or C₆) alkyl-aryl, and O-benzyl;

X_(a) is CR_(a) or N, or N—O;

X_(b) is CR_(b), N, or N—O;

X_(c) is CR_(c) or N, or N—O;

X_(d) is CR_(d) or N, or N—O;

X_(e) is CR_(e), N, or N—O;

R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ are, independently,hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂,C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆)alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂-lower (C₁, C₂,C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl;

P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl is linear or branched alkyl;

K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, aryl,heteroaryl, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉,NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are independently C₁, C₂, C₃, C₄, C₅, or C₆ alkyl orR₁₉ and R₂₀ taken together with the attached nitrogen atom form a fivemembered ring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—;

R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently, H or C₁, C₂,C₃, C₄, C₅, or C₆ alkyl;

Z is (CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, such asbenzene, pyridine, or pyrimidine. For example, Z is:

where R₁, R₂, and R₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆alkyl;

n and m are, independently 0, 1, or 2; R₇, R₈, R₉, R₁₀, and R₁₁ are,independently, hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂H-lower (C₁, C₂,C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl;

P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl is linear or branched alkyl;

K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, aryl,heteroaryl, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are independently C₁, C₂, C₃, C₄, C₅, or C₆ alkyl orR₁₉ and R₂₀ taken together with the attached nitrogen atom form a fivemembered ring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂—, or—OCH₂CH₂CH₂—.

In certain compounds of the invention, Z is

Certain compounds of the invention are selected from Compounds 1-136 and137. For example, the compound of the invention is Compound 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 93, 95,96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137.

Compounds of the invention include Compounds 33, 38, 40, 76, 133, 134,136 and 137.

Certain compounds of the invention are selected from Compounds 138-246and 247. For example, the compound of the invention is Compound 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222,223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, or 247.

Compounds of the invention include Compounds 146 and 147.

Certain compounds of the invention are selected from Compounds 248-273and 274. For example, the compound of the invention is Compound 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, or 274.

In certain Compounds of Formula I, at least one of X_(a), X_(b), X_(c),X_(d) and X_(e) is N.

For example, in the compound of Formula I, X_(a) is N and each of X_(b)is CR_(b), X_(c) is CR_(c), X_(d) is CR_(d) and X_(e) is CR_(e).

In certain compounds of Formula I, X_(y) is CY, and X_(z) is CZ.

For example, in certain compounds of Formula I, Y is hydrogen.

The compounds of the invention can tolerate a wide variety of functionalgroups, so various substituted starting materials can be used tosynthesize them. The syntheses described herein generally provide thedesired final bi-aryl compound at or near the end of the overallprocess, although it may be desirable in certain instances to furtherconvert the compound to a pharmaceutically acceptable salt, ester, orprodrug thereof.

In certain compounds of Formula I, R_(b) is C₁, C₂, C₃, C₄, C₅, or C₆alkoxy. For example, R_(b) is methoxy or ethoxy. In certain compounds ofFormula I, R_(b) is hydrogen. In other compounds of Formula I, R_(b) isselected from F, Cl, Br, and I. For example, R_(b) is F.

In other compounds of Formula I, R_(b) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. For example, V is a bond. In certaincompounds of Formula I, V is —CH₂—, —CH₂CH₂— or —CH₂CH₂CH₂—. In othercompounds, V is —O—CH₂—, —OCH₂CH₂— or —OCH₂CH₂CH₂—.

In certain compounds of Formula I, W is hydrogen. In other compounds, Wis C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In some compounds, W is methyl.

In certain compounds of Formula I, R, is halogen, for example, R, is F,Cl, Br, or I. In some compounds, R_(c) is F. In other compounds, R_(c)is Cl.

In some compounds, R_(c) is C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy. In somecompounds, R_(c) is methoxy or ethoxy. In some embodiments, R_(c) isethoxy.

In other compounds of Formula I, R_(c) is hydrogen.

In other compounds of Formula I, R_(c) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. In some compounds, V is a bond. In othercompounds, V is —CH₂—, —CH₂CH₂— or —CH₂CH₂CH₂—. In other compounds, V is—O—CH₂—, —OCH₂CH₂— or —OCH₂CH₂CH₂—.

In some compounds of Formula I, W is hydrogen. In other compounds, W isC₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In certain compounds, W is methyl.

In certain compounds of Formula I, R_(b) is C₁, C₂, C₃, C₄, C₅, or C₆alkoxy. For example, R_(b) is methoxy or ethoxy. In certain compounds ofFormula I, R_(b) is hydrogen. In other compounds of Formula I, R_(b) isselected from F, Cl, Br, and I. For example, R_(b) is F.

In other compounds of Formula I, R_(b) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. For example, V is a bond. In certaincompounds of Formula I, V is —CH₂—, —CH₂CH₂— or —CH₂CH₂CH₂—. In othercompounds, V is —O—CH₂—, —OCH₂CH₂— or —OCH₂CH₂CH₂—.

In certain compounds of Formula I, W is hydrogen. In other compounds, Wis C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In some compounds, W is methyl.

In certain compounds of Formula I, R_(d) is halogen, for example, R_(d)is F, Cl, Br, or I. In some compounds, R_(d) is F. In other compounds,R_(d) is C₁.

In some compounds, R_(d) is C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy. In somecompounds, R_(d) is methoxy or ethoxy. In some embodiments, R_(d) isethoxy.

In other compounds of Formula I, R_(d) is hydrogen.

In other compounds of Formula I, R_(d) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. In some compounds, V is a bond. In othercompounds, V is —CH₂—, —CH₂CH₂— or —CH₂CH₂CH₂—. In other compounds, V is—O—CH₂—, —OCH₂CH₂— or —OCH₂CH₂CH₂—.

In some compounds of Formula I, W is hydrogen. In other compounds, W isC₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In certain compounds, W is methyl.

The invention relates to a compound of Formula I, having a structureaccording to one of Formulae II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII:

or a salt, solvate, hydrate, or prodrug thereof, where:

R_(b), R₄, R₅, R₈, and R₁₀ are, independently, hydrogen, hydroxyl,halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁,C₂, C₃, C₄, C₅, or C₆ alkyl-OH, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-O-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, COOH, COO-lower (C₁, C₂, C₃, C₄, C₅,or C₆) alkyl, SO₂H, SO₂-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl, and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—.

For example, in the compound of Formula II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII, R₈ is hydrogen, F, Cl, Br, or I. For example,R₈ is F. In certain compounds, R₈ is H.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R_(b) is C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy. Forexample, R_(b) is methoxy or ethoxy. In certain compounds, R_(b) isethoxy. In certain compounds, R_(b) is hydrogen.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R_(b) is Cl, Br, or I. For example, R_(b) is F or C₁.In other compounds, in the compound of Formula II, III, IV, V, VI, VII,VIII, IX, X, XI, XII, and XIII, R_(b) is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl, and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. In some compounds, V is —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—. In certain compounds W is H. In other compounds, W is C₁,C₂, C₃, C₄, C₅, or C₆ alkyl. For example, W is methyl.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R₄ is hydrogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, F,Cl, Br, or I. In some compounds, R₄ is C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy.For example, R₄ is methoxy or ethoxy. In certain compounds, R₄ isethoxy. In other compounds, in the compound of Formula II, III, IV, V,VI, VII, VIII, IX, X, XI, XII, and XIII, R₄ is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. In certain compounds, V is a bond. In othercompounds, V is —CH₂—, —CH₂CH₂— or —CH₂CH₂CH₂—. In other compounds, V is—O—CH₂—, —OCH₂CH₂— or —OCH₂CH₂CH₂—.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R₈ is hydrogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, F,Cl, Br, or I. For example, R₈ is hydrogen. In some compounds, R₈ isethoxy. In certain compounds R₈ is F. In other compounds, in thecompound of Formula II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, andXIII, R₅ is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. In certain compounds, V is a bond. In othercompounds, V is —CH₂—, —CH₂CH₂— or —CH₂CH₂CH₂—. In other compounds, V is—O—CH₂—, —OCH₂CH₂— or —OCH₂CH₂CH₂—.

In certain compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X,XI, XII, and XIII, R₁₀ is hydrogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, F,Cl, Br, or I. In some compounds R₁₀ is C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy.For example, R₁₀ is methoxy or ethoxy. In some compounds, R₁₀ isisobutoxy. In some compounds, R₁₀ is hydrogen. In certain compounds, R₁₀is halogen. For example, R₁₀ is F or C₁.

In other compounds of Formula II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII, R₁₀ is

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; and V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—,—OCH₂CH₂— or —OCH₂CH₂CH₂—. In certain compounds, V is a bond. In othercompounds, V is —CH₂—, —CH₂CH₂— or —CH₂CH₂CH₂—. In other compounds, V is—O—CH₂—, —OCH₂CH₂— or —OCH₂CH₂CH₂—.

For example, in the compound of Formula II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII, W is hydrogen, or C₁, C₂, C₃, C₄, C₅, or C₆alkyl. In some compounds, W is methyl.

Certain compounds of the invention include compounds according toFormula II.

Compounds of the invention include those listed in Table 1:

TABLE 1 Compound # KX # Compound  1 1-136

 2 1-305

 3 1-306

 4 1-307

 5 1-308

 6 1-309

 7 1-310

 8 1-311

 9 1-312

 10 1-313

 11 1-314

 12 1-315

 13 1-316

 14 1-317

 15 1-318

 16 1-319

 17 1-320

 18 1-321

 19 1-322

 20 1-323

 21 1-324

 22 1-325

 23 1-326

 24 1-327

 25 1-329

 26 1-357

 27 1-358

 28 2-359

 29 2-368

 30 2-380

 31 2-378

 32

 33 2-381

 34

 35

 36 2-375

 37 2-386

 38 2-377

 39 2-387

 40 2-365

 41 2-367

 42

 43

 44

 45

 46

 47

 48

 49

 50

 51

 52

 53

 54 2-360

 55 2-369

 56

 57

 58

 59

 60 2-389

 61

 62

 63

 64 2-384

 65

 66 2-388

 67

 68 2-382

 69

 70 2-379

 71

 72 2-373

 73

 74 2-376

 75 2-366

 76 2-361

 77 2-370

 78 2-362

 79 2-363

 80 2-372

 81 2-371

 82 2-364

 83 2-385

 84

 85

 86

 87

 88

 89

 90

 91

 92

 93

 94

 95

 96

 97

 98

 99

100

101

102

103

104

105

106

107

108A 1-072 (Chiral Center)

108B 1-121 (Opposite Enantiomer Of 108A)

109 1-75

110 1-62

111 1-64

112 1-117

113

114 2-390

115 2-374

116 2-383

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133 2-392

134 2-391

135 329-N oxide

136 2-393

137 2-394

Other Compounds are listed in Table 2.

TABLE 2

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

243

244

245

246

247

Compounds of the invention include compounds of Formula IA, and salts,solvates, hydrates, or prodrugs thereof:

wherein: T is absent (i.e., the rings are connected by a bond), CR₁₂R₁₃,C(O), O, S, S(O), S(O)₂, NR₁₄, C(R₁₅R₁₆)C(R₁₇R₁₈), CH₂O, or OCH₂;

X_(y) is CZ, CY, N, or N—O;

X_(z) is CZ, CY, N, or N—O;

at least one of X_(y) and X_(z) is CZ;

Y is selected from hydrogen, hydroxyl, halogen, lower (C₁, C₂, C₃, C₄,C₅, or C₆) alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃,C₄, C₅, or C₆) alkyl-aryl, and O-benzyl;

X_(a) is CR_(a) or N, or N—O;

X_(b) is CR_(b), N, or N—O;

X_(c) is CR_(e) or N, or N—O;

X_(d) is CR_(d) or N, or N—O;

X_(e) is CR_(e), N, or N—O;

R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ are, independently,hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂,C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆)alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂-lower (C₁, C₂,C₃, C₄, C₅, or C₆) alkyl,

wherein W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅,or C₆ alkyl-aryl;

P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl is linear or branched alkyl;

K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NR₁₉R₂₀,SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy, aryl,heteroaryl, or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are independently C₁, C₂, C₃, C₄, C₅, or C₆ alkyl orR₁₉ and R₂₀ taken together with the attached nitrogen atom form a fivemembered ring;

V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—;

R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently, H or C₁, C₂,C₃, C₄, C₅, or C₆ alkyl; and

Z is (CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, R₁, R₂, andR₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; and n and mare, independently 0, 1, or 2;

provided that at least one of R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅,and R₆ is P.

In one embodiment of the invention, at least one of X_(a), X_(b), X_(c),X_(d), X_(e), X_(y) and X_(z) is N. In another embodiment, at least twoof X_(a), X_(b), X_(c), X_(d), X_(e), X_(y) and X_(z) are N. In anotherembodiment, at least one of X_(a) and X_(y) is N. For example, bothX_(a) and X_(y) are N. In another embodiment, X_(a), X_(b), X_(c),X_(d), and X_(e) are not each N or N—O. In another embodiment, X_(c),X_(d), and X_(e) are not each N or N—O.

In one embodiment, T is absent e.g., a bond. In another embodiment,X_(b) is CR_(b). In another embodiment, R_(b) is P. For example, in oneembodiment, P is O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K. In oneembodiment, lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl is CH₂CH₂CH₂. In oneembodiment, lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl is branched alkyl.For example, branched alkyl is

In another embodiment, K, L, M, or Q, if present, is lower C₁, C₂, C₃,C₄, C₅ or C₆ alkoxy. For example, K is methoxy. In one embodiment,branched alkyl is

and K is methoxy. In another embodiment, K, L, M, or Q, if present, isCOOH. For example, in one embodiment, K is COOH. In another embodiment,K, L, M, or Q, if present, is aryl or heteroaryl. For example,heteroaryl is tetrazole.

In one embodiment, R_(b) is

In another embodiment, R_(b) is

In one embodiment, V is —OCH₂CH₂. In another embodiment, V is a bond. Inone embodiment, W is C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. For example, W ismethyl or ethyl.

In one embodiment, X_(z) is CZ, further wherein Z is

and R₇, R₈, R₉, R₁₀, and R₁₁ are selected from hydrogen, hydroxyl,halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁,C₂, C₃, C₄, C₅, or C₆ alkyl-OH, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-O—C₁,C₂, C₃, C₄, C₅, or C₆ alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl. In another embodiment, at least one of R₇, R₈, R₉, R₁₀,and R₁₁ is halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, or O-benzyl. Inanother embodiment, at least one of R₈ or R₁₀ is halogen. For example,halogen is fluorine. In another embodiment, at least one of R₇ or R₁₁ isC₁, C₂, C₃, C₄, C₅, or C₆ alkoxy or O-benzyl. For example, at least oneof R₇ or R₁₁ is ethoxy or at least one of R₇ or R₁₁ is O-benzyl. In oneembodiment, R₁₁ is H. In one embodiment, n is 1. In one embodiment, R₂is H. In one embodiment, R₃ is H. In one embodiment, m is 1. In anotherembodiment, m and n are each 1 and R₂ and R₃ are each H.

In one embodiment, R₄ and R₆ are each H. In another embodiment R₅ isselected from halogen and C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. In oneembodiment, R₅ is halogen. For example, R₅ is Cl or F. In anotherembodiment, R₅ is C₁, C₂, C₃, C₄, C₅, or C₆ alkyl. For example, R₅ ismethyl or ethyl.

The invention includes a solvate of a compound according to Formula IA.The invention includes a hydrate of compound according to Formula IA.The invention includes an acid addition salt of a compound according toFormula IA. For example, a hydrochloride salt. In another embodiment,the invention includes a pharmaceutically acceptable salt. The inventionincludes a composition comprising a compound of Formula IA and at leastone pharmaceutically acceptable excipient.

Certain compounds of the invention include compounds selected from Table3.

TABLE 3 Compound # Structure 248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

The invention relates to a solvate of a compound according to one ofFormulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, orXII. The invention also relates to a hydrate of a compound according toone of Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, or XIII.

The invention also relates to an acid addition salt of a compoundaccording to one of Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, or XIII. For example, a hydrochloride salt e.g., adihydrochloride salt.

Further, the invention relates to a prodrug of a compound according toone of Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, or XIII.

The invention also relates to a pharmaceutically acceptable salt of acompound of one of Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, or XIIII.

The invention includes compositions comprising a compound according toone of Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, or XIII and at least one pharmaceutically acceptable excipient.

Certain compounds of the invention are non-ATP competitive kinaseinhibitors.

The invention also includes a method of preventing or treating a cellproliferation disorder by administering a pharmaceutical compositionthat includes a compound according to one of Formulae I, IA, IB, II,III, IV, V, VI, VII, VIII, IX, X, XI, XII, or XIII, or a salt, solvate,hydrate, or prodrug thereof, and at least one pharmaceuticallyacceptable excipient to a subject in need thereof. The invention alsoincludes use of a compound of the invention in the manufacture of amedicament to prevent or treat a cell proliferation disorder.

For example, the cell proliferation disorder is pre-cancer or cancer.The cell proliferation disorder treated or prevented by the compounds ofthe invention may be a cancer, such as, for example, colon cancer orlung cancer.

The cell proliferation disorder treated or prevented by the compounds ofthe invention may be a hyperproliferative disorder.

The cell proliferation disorder treated or prevented by the compounds ofthe invention may be psoriases.

For example, the treatment or prevention of the proliferative disordermay occur through the inhibition of a tyrosine kinase. For example, thetyrosine kinase can be a Src kinase or focal adhesion kinase (FAK).

The invention relates to a method of treating or preventing a disease ordisorder that is modulated by kinase inhibition, by administering apharmaceutical composition that includes a compound according to FormulaI, IA or IB or one of Formulae II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, or XIII, or a salt, solvate, hydrate, or prodrug thereof, and atleast one pharmaceutically acceptable excipient. For example, thedisease or disorder that is modulated by tyrosine kinase inhibition iscancer, pre-cancer, a hyperproliferative disorder, or a microbialinfection. For example, the compound is a compound according to FormulaI, IA, IB or II.

The pharmaceutical composition of the invention may modulate a kinasepathway. For example, the kinase pathway is a Src kinase pathway, orfocal adhesion kinase pathway.

The pharmaceutical composition of the invention may modulate a kinasedirectly. For example, the kinase is Src kinase, or focal adhesionkinase.

Certain pharmaceutical compositions of the invention are non-ATPcompetitive kinase inhibitors.

For example, the compounds of the invention are useful to treat orprevent a microbial infection, such as a bacterial, fungal, parasitic orviral infection. The invention also includes use of a compound of theinvention in the manufacture of a medicament to prevent or treat amicrobial infection.

Certain pharmaceutical compositions of the invention include a compoundselected from Compound 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, and 137. For example, the pharmaceuticalcomposition includes Compound 33, 38, 40, 76, 133, 134, 136 or 137.

Certain pharmaceutical compositions of the invention include a compoundselected from Compound 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, or 247. For example, the pharmaceutical composition includesCompound 146 or 147.

Certain pharmaceutical compositions of the invention include a compoundselected from Compounds 248-274. For example, the compound of theinvention is Compound 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, or 274.

A compound of the invention may be used as a pharmaceutical agent. Forexample, a compound of the invention is used as an anti-proliferativeagent, for treating humans and/or animals, such as for treating humansand/or other mammals. The compounds may be used without limitation, forexample, as anti-cancer, anti-angiogenesis, anti-microbial,anti-bacterial, anti-fungal, anti-parasitic and/or anti-viral agents.Additionally, the compounds may be used for other cellproliferation-related disorders such as diabetic retinopathy, maculardegeneration and psoriases. Anti-cancer agents include anti-metastaticagents.

The compound of the invention used as a pharmaceutical agent may beselected from Compounds 1-136 and 137. For example, the compound of theinvention used as a pharmaceutical agent is Compound 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 93, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137. For example,the compound of the invention used as a pharmaceutical agent is selectedfrom Compounds 33, 38, 40, 76, 133, 134, 136 and 137.

Certain pharmaceutical agents include a compound selected from theCompound 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, or 247.For example, the compound of the invention used as a pharmaceuticalagent is selected from Compounds 146 and 147.

Certain pharmaceutical agents include a compound selected from Compounds248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, or 274.

In one aspect of the invention, a compound of the invention, forexample, a compound of Formula I, IA or IB, or one of Formulae II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, or XIII, is used to treat orprevent a cell proliferation disorder in an subject. In one aspect ofthe embodiment, the cell proliferation disorder is pre-cancer or cancer.In another aspect of the embodiment, the cell proliferation disorder isa hyperproliferative disorder. In another embodiment, prevention ortreatment of the cell proliferation disorder, cancer orhyperproliferative disorder occurs through the inhibition of a kinase.In another embodiment, prevention or treatment of the cell proliferationdisorder, cancer or hyperproliferative disorder occurs through theinhibition of a tyrosine kinase. In another embodiment, prevention ortreatment of the cell proliferation disorder, cancer orhyperproliferative disorder occurs through the inhibition of Src kinaseor focal adhesion kinase (FAK). In another embodiment, the subject is amammal. In one embodiment, the subject is human.

Another aspect of the invention includes a method of protecting againstor treating hearing loss in a subject comprising administering acompound having the Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, and XIII. The invention also includes use of a compoundof the invention in the manufacture of a medicament to protect againstor treat hearing loss.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In one embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound does not inhibitATP binding to the protein kinase. In one embodiment, the compoundinhibits a Src family protein kinase. In one embodiment, the Src familyprotein kinase is pp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically e.g., by administering drops intothe ear, intraarterially, intralesionally, by metering pump, or byapplication to mucous membranes. In another embodiment, the compound isadministered with a pharmaceutically acceptable carrier.

In one embodiment, the compound is administered before initiation ofhearing loss. In another embodiment, the compound is administered afterinitiation of hearing loss.

In one embodiment, the compound is administered in combination with adrug that causes hearing loss e.g., cis platinum or an aminoglycosideantibiotic. In another embodiment, the compound is administered incombination with a drug that targets hairy cells.

In one embodiment, at least one of X_(a), X_(b), X_(c), X_(d), X_(e),X_(y) and X_(z) is N. In another embodiment, T is absent e.g., a bond.In another embodiment, X_(z) is CZ and Z is

In one embodiment, m and n are each 1 and R₂ and R₃ are each H. Inanother embodiment, at least one of R₇, R₈, R₉, R₁₀ and R₁₁ is halogen,C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, or O-benzyl. In one embodiment, thecompound is Compound 25 (KX1-329).

Another aspect of the invention includes a method of protecting againstor treating osteoporosis in a subject comprising administering acompound having a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX,X, XI, XII, and XIII. The invention also includes use of a compound ofthe invention in the manufacture of a medicament to prevent or treatosteoporosis.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, at least one of X_(a), X_(b), X_(c), X_(d), X_(e),X_(y) and X_(z) is N. In another embodiment, T is absent e.g., a bond.In another embodiment, X_(z) is CZ and Z is

In one embodiment, m and n are each 1 and R₂ and R₃ are each H. Inanother embodiment, at least one of R₇, R₈, R₉, R₁₀ and R₁₁ is halogen,C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, or O-benzyl. In one embodiment, thecompound is Compound 25 (KX1-329), Compound 38 (KX2-377), Compound 76(KX2-361), Compound 133 (KX2-392), Compound 134 (KX2-391), or Compound137 (KX2-394).

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore initiation of osteoporosis. In another embodiment, the compoundis administered after initiation of osteoporosis.

Another aspect of the invention includes a method of protecting againstor treating ophthalmic diseases e.g., macular degeneration, retinopathy,macular edema, etc. in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. The invention also includes use of a compound of theinvention in the manufacture of a medicament to prevent or treatophthalmic diseases.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase. In another embodiment, the compoundinhibits one or more components in the VEGF pathway.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically (e.g., by administering drops tothe eye), intraarterially, intralesionally, by metering pump, or byapplication to mucous membranes. In one embodiment, the compound isadministered with a pharmaceutically acceptable carrier. In oneembodiment, the compound is administered before initiation of theophthalmic disease. In another embodiment, the compound is administeredafter initiation of ophthalmic disease.

Another aspect of the invention includes a method of protecting againstor treating diabetes in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. The invention also includes use of a compound of theinvention in the manufacture of a medicament to protect against or treatdiabetes.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore initiation of the diabetes. In another embodiment, the compoundis administered after initiation of disease.

Another aspect of the invention includes a method of protecting againstor treating obesity in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. The invention also includes use of a compound of theinvention in the manufacture of a medicament to protect against or treatobesity.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore the subject is obese. In another embodiment, the compound isadministered after the subject is obese.

Another aspect of the invention includes a method of protecting againstor treating stroke in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. The invention also includes use of a compound of theinvention in the manufacture of a medicament to protect against or treatstroke.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60-src tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore a stroke has occurred. In another embodiment, the compound isadministered after a stroke has occurred.

Another aspect of the invention includes a method of protecting againstor treating athrosclerosis in a subject comprising administering acompound having a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX,X, XI, XII, and XIII. The invention also includes use of a compound ofthe invention in the manufacture of a medicament to protect against ortreat athrosclerosis.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier.

Another aspect of the invention includes a method of regulating immunesystem activity in a subject comprising administering a compound havinga Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, andXIII. The invention also includes use of a compound of the invention inthe manufacture of a medicament to regulate immune system activity.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier.

Another aspect of the invention includes a method of protecting againstor treating hepatitis B in a subject comprising administering a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII. The invention also includes use of a compound of theinvention in the manufacture of a medicament to protect against or treathepatitis B.

In one embodiment, the compound inhibits one or more components of akinase signaling cascade. In another embodiment, the compound is anallosteric inhibitor. In one embodiment, the compound is a peptidesubstrate inhibitor. In one embodiment, the compound inhibits a Srcfamily protein kinase. For example, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier. In one embodiment, the compound is administeredbefore the subject has contracted hepatitis B. In another embodiment,the compound is administered after the subject has contracted hepatitisB.

Another aspect of the invention is a method of preventing or treating acell proliferation disorder comprising administering to a subject inneed thereof a compound having the Formula IA. In one embodiment, thecompound inhibits one or more components of a protein kinase signalingcascade. In another embodiment, the compound is an allosteric inhibitor.In another embodiment, the compound is a peptide substrate inhibitor. Inanother embodiment, the compound does not inhibit ATP binding to aprotein kinase. In one embodiment, the compound inhibits a Src familyprotein kinase. In another embodiment, the Src family protein kinase ispp60^(c-src) tyrosine kinase.

In one embodiment, at least one of X_(a), X_(b), X_(c), X_(d), X_(e),X_(y) and X_(z) is N. In another embodiment, X_(z) is CZ, furtherwherein Z is

and R₇, R₈, R₉, R₁₀, and R₁₁ are selected from hydrogen, hydroxyl,halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, O-benzyl, C₁,C₂, C₃, C₄, C₅, or C₆ alkyl-OH, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-O—C₁,C₂, C₃, C₄, C₅, or C₆ alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl. In one embodiment, at least one of R₇, R₈, R₉, R₁₀, andR₁₁ is halogen, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, or O-benzyl. Inanother embodiment, m and n are each 1 and R₂ and R₃ are each H. In oneembodiment, R₄ and R₆ are each H. In one embodiment of the invention, acompound is selected from 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, or 274.

The invention is also drawn to a method of treating or preventing canceror a proliferation disorder in a subject, comprising administering aneffective amount of a compound of the invention, for example, a compoundof Formula I, IA, IB, or one of Formulae II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, or XIII. For example, the compound of the invention maybe a kinase inhibitor. The compound of the invention may be a non-ATPcompetitive kinase inhibitor. The compound of the invention may inhibita kinase directly, or it may affect the kinase pathway.

In certain embodiments, the cell proliferation disorder includes anytype of cancer including solid tumors and non-solid tumors. In specificembodiments the solid tumors are selected from tumors in the CNS(central nervous system), liver cancer, colorectal carcinoma, breastcancer, gastric cancer, pancreatic cancer, bladder carcinoma, cervicalcarcinoma, head and neck tumors, vulvar cancer and dermatologicalneoplasms including melanoma, squamous cell carcinoma and basal cellcarcinomas. In other embodiment, non-solid tumors includelymphoproliferative disorders including leukemias and lymphomas. Inother embodiments a disorder is metastatic disease.

The compounds of the present invention display broad solid tumoractivity, as is reported in Table 4 below.

TABLE 4 KXO1 GI50 Dasatinib GI50 Human Tumor Cell Line (nM) (nM) HT29(Colon) 25   20    SKOV-3 (Ovarian) 9.8 3.2 PC3-MM2 (Prostate) 8.9 8.9L3.6pl (Pancreas) 25 (n = 3) 3.9 MDA231 (Breast) 20   6.9 A549 (Lung)9.4 13  

The compound of the present invention also may be used in the treatmentof a cancer or cell proliferation disorder in combination therapy withone or more of anti-cancer treatments such as radiation therapy, and/orone or more anti-cancer agents selected from the group consisting ofanti-proliferative agents, cytotoxic agents, cytostatic agents, andchemotherapeutic agents and salts and derivatives thereof. According tocertain embodiments, the compound of the present invention may be usedin the treatment of a cancer or cell proliferation disorder incombination therapy with any one of the drugs selected from a groupconsisting of an alkaloid, an alkylating agent, an antitumor antibiotic,an antimetabolite, an Bcr-Abl tyrosine kinase inhibitor, a nucleosideanalogue, a multidrug resistance reversing agent, a DNA binding agent,microtubule binding drug, a toxin and a DNA antagonist. Those of skillin the art will recognize the chemotherapeutic agents classified intoone or more particular classes of chemotherapeutic agents describedabove.

According to preferred embodiments, the compound of the presentinvention may be used in the treatment of a cancer or cell proliferationdisorder in combination therapy with one or more agents selected fromthe group consisting of antimetabolites (e.g., gemcitabine), inhibitorsof topoisomerase I and II, alkylating agents and microtubule inhibitors(e.g., taxol), as well as tyrosine kinase inhibitors (e.g., surafenib),EGF kinase inhibitors (e.g., tarceva or erlotinib), platinum complexes(e.g., oxaliplatin); and ABL kinase inhibitors (e.g., Gleevec orImatinib).

Alkaloids include, but are not limited to, docetaxel, etoposide,irinotecan, paclitaxel (Taxol), teniposide, topotecan, vinblastine,vincristine, vindesine.

Alkylating agents include, but are not limited to, busulfan,improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa,altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, chlorambucil, chloranaphazine,cyclophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide HCl, melphalan novemebichin, perfosfamidephenesterine, prednimustine, trofosfamide, uracil mustard, carmustine,chlorozotocin, fotemustine, lomustine, nimustine, semustine ranimustine,dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,temozolomide.

Antibiotics and analogs thereof include, but are not limited to,aclacinomycins, actinomycins, anthramycin, azaserine, bleomycins,cactinomycin, carubicin, carzinophilin, cromomycins, dactinomycins,daunorubicin, 6-diazo-5-oxo-1-norleucine, doxorubicin, epirubicin,idarubicin, menogaril, mitomycins, mycophenolic acid, nogalamycine,olivomycins, peplomycin, pirarubicin, plicamycin, porfiromycin,puromycine, streptonigrin, streptozocin, tubercidin, zinostatin,zorubicin.

Antimetabolites include, but are not limited to, denopterin, edatrexate,mercaptopurine (6-MP), methotrexate, piritrexim, pteropterin,pentostatin (2′-DCF), tomudex, trimetrexate, cladridine, fludarabine,thiamiprine, ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, doxifluridine, emitefur, floxuridine, fluorouracil,gemcitabine, tegafur, hydroxyurea and urethan.

Platinum complexes include, but are not limited to, caroplatin,cisplatin, miboplatin, oxaliplatin.

Anti-mitotic agents or microtubule binding agents include, but are notlimited to, vincristine, and vinblastine, and taxol.

When use in combination with additional anti-proliferation agents, thecompounds of the present invention may enhance (e.g., synergize) theactivity of these agents. Further, such synergism would permit thecompounds of the present invention, additional anti-proliferationagents, or both to be administered at lower dosages, and/or maysignificantly enhance the anti-proliferation properties of the compoundsat any given dose. Table 5 provides the results of combinationtreatments using the compounds of the present invention and additionalanti-proliferation agents.

TABLE 5 Drug1 + KXO1 Drug 1-GI₅₀ KXO1-GI₅₀ Drug1:KXO1 Combo GI₅₀ CellLine (nM) (nM) GI₅₀ ratio (nM) Result HT29 1,480 (n = 2) 25 (n = 5) 59 180 + 1.8  Synergy, (Colon) oxaliplatin (used 100 X) ca.10 X SKOV-3 3.9 (n = 2) 9.8 (n = 1)  0.40 3.9 + 11  No (Ovarian) taxol interferenceA549 (Lung) 1,735 (n = 2) 13 (n = 3) 134 2,500 + 11   No Tarceva (used233 X) interference L3.6pl  2.0 (n = 2) 32 (n = 4) 1/13 0.09 + 1.15Synergy, (Pancreas) Gemcitabine ca. 25 X

According to another embodiment, there is provided a method for treatingleukemia in a host comprising administering to a patient a compoundhaving a Formulae I, IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII.

In another embodiment, there is provided a method for treating leukemiain a host comprising administering to a patient a therapeuticallyeffective amount of a compound according to Formulae I, IA, IB, II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII, as defined above, and atleast one further therapeutic agent selected from the group consistingof anti-proliferative agents, cytotoxic agents, cytostatic agents, andchemotherapeutic agents and salts and derivatives thereof. According tocertain embodiments, the compound of the present invention may be usedin the treatment of a leukemia in combination therapy with one or moreof the drugs selected from a group consisting of an alkaloid, analkylating agent, an antitumor antibiotic, an antimetabolite, an Bcr-Abltyrosine kinase inhibitor, a nucleoside analogue, a multidrug resistancereversing agent, a DNA binding agent, microtubule binding drug, a toxinand a DNA antagonist. Those of skill in the art will recognize thechemotherapeutic agents classified into one or more particular classesof drugs described above.

Leukemia is a malignant cancer of the bone marrow and blood and ischaracterized by the uncontrolled growth of blood cells. The commontypes of leukemia are divided into four categories: acute or chronicmyelogenous, involving the myeloid elements of the bone marrow (whitecells, red cells, megakaryocytes) and acute or chronic lymphocytic,involving the cells of the lymphoid lineage. Treatment of leukemiagenerally depends upon the type of leukemia. Standard treatment forleukemia usually involves chemotherapy and/or bone marrowtransplantation and/or radiation therapy. See e.g., U.S. Pat. No.6,645,972, hereby incorporated herein by reference in its entirety.

Chemotherapy in leukemia may involve a combination of two or moreanti-cancer drugs. Approximately 40 different drugs are now being usedin the treatment of leukemia, either alone or in combination. Othertreatments for leukemia also include the reversal of multidrugresistance, involving the use of agents which decrease the mechanismsallowing the malignant cells to escape the damaging effects of thechemotherapeutic agent (and leads to refractoriness or relapses); andbiological therapy, involving the use of monoclonal antibodies, in whichtoxins are attached to antibodies that react with the complementaryantigen carried by the malignant cells; and cytokines (e.g.,interferons, interleukins, colony-stimulating factors CSFs) which arenaturally occurring chemicals that stimulate blood cell production andhelp restore blood cell counts more rapidly after treatment. Examples ofthese drugs include multidrug resistance reversing agent PSC 833, themonoclonal antibody Rituxan and the following cytokines: Erythropoetinand Epoetin, which stimulate the production of red cells; G-CSF, GM-CSF,filgrastim, and Sargramostim which stimulate the production of whitecells; and thrombopoietin, which stimulate the production of platelets.

Many nucleoside analogues have been found to possess anticanceractivity. Cytarabine, Fludarabine, Gemcitabine and Cladribine are someexamples of nucleoside analogues which are currently important drugs inthe treatment of leukemia. β-L-OddC ((−)-β-L-Dioxolane-Cytidine,Troxatyl®, from Shire BioChem Inc.) is also a nucleoside analogue whichwas first described as an antiviral agent by Belleau et al. (EP 337713,herein incorporated by reference in its entirety) and was shown to havepotent antitumor activity (K. L. Grove et al., Cancer Res., 55(14),3008-11, 1995; K. L. Grove et al., Cancer Res., 56(18), 4187-4191, 1996,K. L. Grove et al., Nucleosides Nucleotides, 16:1229-33, 1997; S. AKadhim et al., Can. Cancer Res., 57(21), 4803-10, 1997). In clinicalstudies, O-L-OddC has been reported to have significant activity inpatients with advanced leukemia (Giles et al., J. Clin. Oncology, Vol19, No 3, 2001).

Bcr-Abl tyrosine kinase inhibitors, such as STI-571 (Gleevec®, Imatinibmesylate, from Novartis Pharmaceuticals Corp.), have shown significantantileukemic activity and specifically in chronic myeologenous leukemia.STI-571, for example, has become a promising therapy in the group ofpatients targeting Bcr-Abl tyrosine kinase inhibition. However, despitesignificant hematologic and cytogenic responses, resistance to Bcr-Abltyrosine kinase inhibitors occurs, particularly in the advanced phasesof chronic myelogenous leukemia. Such resistance has been demonstratedfor the Bcr-Abl tyrosine kinase inhibitors Imatinib, Dasatinib, AZD0530.

Accordingly, there is a great need for the further development of agentsfor the treatment of leukemia patients who have been previously treatedwith a Bcr-Abl tyrosine kinase inhibitor and have become resistant tothe Bcr-Abl tyrosine kinase inhibitor. Thus, in another embodiment ofthe present invention, there is provide a method for treating leukemiain a host comprising administering to a patient that has been previouslytreated with a Bcr-Abl tyrosine kinase inhibitor and has becomeresistant to the Bcr-Abl tyrosine kinase inhibitor treatment, atherapeutically effective amount of a compound according to Formulae I,IA, IB, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, and XIII.Further, there is provide a method for combination therapy of leukemiain a host comprising administering to a patient a Bcr-Abl tyrosinekinase inhibitor in combination with a therapeutically effective amountof a compound according to Formulae I, IA, IB, II, III, IV, V, VI, VII,VIII, IX, X, XI, XII, and XIII. In an preferred embodiment, thecombination is administered to a patient that has become resistant tothe Bcr-Abl tyrosine kinase inhibitor treatment.

The compounds of the present invention display anti-leukemia activity ascompared to existing therapeutic agents, as is show in Table 6 below.

TABLE 6 Human Leukemia KXO1 GI50 Dasatinib Cell Line (nM) GI50 (nM) K562(CML) 13 (n = 2)  0.37 (n = 1-2) K562R (Gleevec resistant 0.64 (n = 1-2)0.81 (n = 2)  CML) MOLT-4 (Adult 13 (n = 1) 644 (n = 1) lymphoblasticleukemia) CCRF-HSB-2 (Adult 12 (n = 1) Inactive (n = 1) lymphoblasticleukemia) Jurkat (Adult T cell 10 (n = 1)  8 (n = 1) leukemia) Ba/F3(IL-3 induced) 3.5 Inactive Ba/F3 + WT BCR-Abl 85     1 Ba/F3 + BCR-AblE225K 80     1 mutant Ba/F3 + BCR-Abl T315I 35 >10,000 mutant

In one embodiment, the administration of the compound is carried outorally, parentally, subcutaneously, intravenously, intramuscularly,intraperitoneally, by intranasal instillation, by intracavitary orintravesical instillation, topically, intraarterially, intralesionally,by metering pump, or by application to mucous membranes. In oneembodiment, the compound is administered with a pharmaceuticallyacceptable carrier.

Definitions

For convenience, certain terms used in the specification, examples andappended claims are collected here.

Protein kinases are a large class of enzymes which catalyze the transferof the γ-phosphate from ATP to the hydroxyl group on the side chain ofSer/Thr or Tyr in proteins and peptides and are intimately involved inthe control of various important cell functions, perhaps most notably:signal transduction, differentiation, and proliferation. There areestimated to be about 2,000 distinct protein kinases in the human body,and although each of these phosphorylate particular protein/peptidesubstrates, they all bind the same second substrate ATP in a highlyconserved pocket. About 50% of the known oncogene products are proteintyrosine kinases (PTKs), and their kinase activity has been shown tolead to cell transformation.

The PTKs can be classified into two categories, the membrane receptorPTKs (e.g. growth factor receptor PTKs) and the non-receptor PTKs (e.g.the Src family of protooncogene products and focal adhesion kinase(FAK)). The hyperactivation of Src has been reported in a number ofhuman cancers, including those of the colon, breast, lung, bladder, andskin, as well as in gastric cancer, hairy cell leukemia, andneuroblastoma.

The phrase “inhibits one or more components of a protein kinasesignaling cascade” means that one or more components of the kinasesignaling cascade are effected such that the functioning of the cellchanges. Components of a protein kinase signaling cascade include anyproteins involved directly or indirectly in the kinase signaling pathwayincluding second messengers and upstream and downstream targets.

“Treating”, includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder, etc. “Treating” or “treatment” of a disease stateincludes: (a) inhibiting an existing disease-state i.e., arresting itsdevelopment or clinical symptoms; and/or (b) relieving the disease-statei.e., causing regression of the disease.

“Preventing” means causing the clinical symptoms of the disease statenot to develop i.e., inhibiting the onset of disease, in a subject thatmay be exposed to or predisposed to the disease state, but does not yetexperience or display symptoms of the disease state.

“Disease state” means any disease, disorder, condition, symptom, orindication.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the terms “psoriatic condition” or “psoriasis” refers todisorders involving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration.

In one embodiment, the cell proliferation disorder is cancer. As usedherein, the term “cancer” includes solid tumors, such as lung, breast,colon, ovarian, brain, liver, pancreas, prostate, malignant melanoma,non-melanoma skin cancers, as well as hematologic tumors and/ormalignancies, such as childhood leukemia and lymphomas, multiplemyeloma, Hodgkin's disease, lymphomas of lymphocytic and cutaneousorigin, acute and chronic leukemia such as acute lymphoblastic, acutemyelocytic or chronic myelocytic leukemia, plasma cell neoplasm,lymphoid neoplasm and cancers associated with AIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses andthe like. The proliferative diseases can include dysplasias anddisorders of the like.

An “effective amount” of a compound of the disclosed invention is thequantity which, when administered to a subject having a disease ordisorder, results in regression of the disease or disorder in thesubject. Thus, an effective amount of a compound of the disclosedinvention is the quantity which, when administered to a subject having acell proliferation disorder, results in regression of cell growth in thesubject. The amount of the disclosed compound to be administered to asubject will depend on the particular disorder, the mode ofadministration, co-administered compounds, if any, and thecharacteristics of the subject, such as general health, other diseases,age, sex, genotype, body weight and tolerance to drugs. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors.

As used herein, the term “effective amount” refers to an amount of acompound, or a combination of compounds, of the present inventioneffective when administered alone or in combination as ananti-proliferative agent. For example, an effective amount refers to anamount of the compound present in a formulation or on a medical devicegiven to a recipient patient or subject sufficient to elicit biologicalactivity, for example, anti-proliferative activity, such as e.g.,anti-cancer activity or anti-neoplastic activity. The combination ofcompounds optionally is a synergistic combination. Synergy, asdescribed, for example, by Chou and Talalay, Adv. Enzyme Regul. vol. 22,pp. 27-55 (1984), occurs when the effect of the compounds whenadministered in combination is greater than the additive effect of thecompounds when administered alone as a single agent. In general, asynergistic effect is most clearly demonstrated at sub-optimalconcentrations of the compounds. Synergy can be in terms of lowercytotoxicity, or increased anti-proliferative effect, or some otherbeneficial effect of the combination compared with the individualcomponents.

“A therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

A therapeutically effective amount of one or more of the compounds canbe formulated with a pharmaceutically acceptable carrier foradministration to a human or an animal. Accordingly, the compounds orthe formulations can be administered, for example, via oral, parenteral,or topical routes, to provide an effective amount of the compound. Inalternative embodiments, the compounds prepared in accordance with thepresent invention can be used to coat or impregnate a medical device,e.g., a stent.

The term “prophylactically effective amount” means an effective amountof a compound or compounds, of the present invention that isadministered to prevent or reduce the risk of unwanted cellularproliferation.

“Pharmacological effect” as used herein encompasses effects produced inthe subject that achieve the intended purpose of a therapy. In oneembodiment, a pharmacological effect means that primary indications ofthe subject being treated are prevented, alleviated, or reduced. Forexample, a pharmacological effect would be one that results in theprevention, alleviation or reduction of primary indications in a treatedsubject. In another embodiment, a pharmacological effect means thatdisorders or symptoms of the primary indications of the subject beingtreated are prevented, alleviated, or reduced. For example, apharmacological effect would be one that results in the prevention orreduction of primary indications in a treated subject.

With respect to the chemical compounds useful in the present invention,the following terms can be applicable:

The term “substituted,” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced. Keto substituents are not present on aromatic moieties. Ringdouble bonds, as used herein, are double bonds that are formed betweentwo adjacent ring atoms (e.g., C═C, C═N, or N═N).

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Alltautomers of shown or described compounds are also considered to be partof the present invention.

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R₁ moieties,then the group may optionally be substituted with up to two R₁ moietiesand R¹ at each occurrence is selected independently from the definitionof R₁. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

Compounds of the present invention that contain nitrogens can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (m-CPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₆ alkynyl,C₃₋₁₄ carbocycle, or 3-14-membered heterocycle) derivatives.

When an atom or chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), the invention is meant to encompass each numberwithin the range as well as all intermediate ranges. For example, “C₁₋₆alkyl” is meant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5,1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6carbons.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms. For example, C₁₋₆ alkyl is intended toinclude C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. Examples of alkylinclude, but are not limited to, methyl, ethyl, n-propyl, i-propyl,n-butyl, s-butyl, t-butyl, n-pentyl, s-pentyl, and n-hexyl. “Alkyl”further includes alkyl groups that have oxygen, nitrogen, sulfur orphosphorous atoms replacing one or more hydrocarbon backbone carbonatoms. In certain embodiments, a straight chain or branched chain alkylhas six or fewer carbon atoms in its backbone (e.g., C₁-C₆ for straightchain, C₃-C₆ for branched chain), and in another embodiment, a straightchain or branched chain alkyl has four or fewer carbon atoms. Likewise,cycloalkyls have from three to eight carbon atoms in their ringstructure, and in another embodiment, cycloalkyls have five or sixcarbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl”includes an alkyl group, as defined above, but having from one to ten,or in another embodiment from one to six, carbon atoms in its backbonestructure. “Lower alkenyl” and “lower alkynyl” have chain lengths of,for example, 2-5 carbon atoms.

The term “substituted alkyls” refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includesstraight-chain alkenyl groups (e.g., ethenyl, propenyl, butenyl,pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl), branched-chainalkenyl groups, cycloalkenyl (e.g., alicyclic) groups (e.g.,cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, andcycloalkyl or cycloalkenyl substituted alkenyl groups. The term“alkenyl” further includes alkenyl groups, which include oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more hydrocarbonbackbone carbons. In certain embodiments, a straight chain or branchedchain alkenyl group has six or fewer carbon atoms in its backbone (e.g.,C₂-C₆ for straight chain, C₃-C₆ for branched chain). Likewise,cycloalkenyl groups may have from three to eight carbon atoms in theirring structure, and in one embodiment, cycloalkenyl groups have five orsix carbons in the ring structure. The term “C₂-C₆” includes alkenylgroups containing two to six carbon atoms. The term “C₃-C₆” includesalkenyl groups containing three to six carbon atoms.

The term “substituted alkenyls” refers to alkenyl moieties havingsubstituents replacing a hydrogen on one or more hydrocarbon backbonecarbon atoms. Such substituents can include, for example, alkyl groups,alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight-chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), branched-chain alkynyl groups, andcycloalkyl or cycloalkenyl substituted alkynyl groups. The term“alkynyl” further includes alkynyl groups having oxygen, nitrogen,sulfur or phosphorous atoms replacing one or more hydrocarbon backbonecarbons. In certain embodiments, a straight chain or branched chainalkynyl group has six or fewer carbon atoms in its backbone (e.g., C₂-C₆for straight chain, C₃-C₆ for branched chain). The term “C₂-C₆” includesalkynyl groups containing two to six carbon atoms. The term “C₃-C₆”includes alkynyl groups containing three to six carbon atoms.

The term “substituted alkynyls” refers to alkynyl moieties havingsubstituents replacing a hydrogen on one or more hydrocarbon backbonecarbon atoms. Such substituents can include, for example, alkyl groups,alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aryl” includes groups with aromaticity, including 5- and 6-membered“unconjugated”, or single-ring, aromatic groups that may include fromzero to four heteroatoms, as well as “conjugated”, or multicyclic,systems with at least one aromatic ring. Examples of aryl groups includebenzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiazole,imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine,pyrazine, pyridazine, and pyrimidine, and the like. Furthermore, theterm “aryl” includes multicyclic aryl groups, e.g., tricyclic, bicyclic,e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole,benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline,isoquinoline, napthridine, indole, benzofuran, purine, benzofuran,deazapurine, or indolizine. Those aryl groups having heteroatoms in thering structure may also be referred to as “aryl heterocycles”,“heterocycles,” “heteroaryls” or “heteroaromatics”. The aromatic ringcan be substituted at one or more ring positions with such substituentsas described above, as for example, halogen, hydroxyl, alkoxy,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminocarbonyl,aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl,aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino(including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Arylgroups can also be fused or bridged with alicyclic or heterocyclicrings, which are not aromatic so as to form a multicyclic system (e.g.,tetralin, methylenedioxyphenyl).

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo, andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

“Counterion” is used to represent a small, negatively charged speciessuch as chloride, bromide, hydroxide, acetate, and sulfate.

The term “non-hydrogen substituent” refers to substituents other thanhydrogen. Non-limiting examples include alkyl groups, alkoxy groups,halogen groups, hydroxyl groups, aryl groups, etc.

As used herein, “carbocycle” or “carbocyclic ring” is intended to meanany stable monocyclic, bicyclic, or tricyclic ring having the specifiednumber of carbons, any of which may be saturated, unsaturated, oraromatic. For example a C₃₋₁₄ carbocycle is intended to mean a mono-,bi-, or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14carbon atoms. Examples of carbocycles include, but are not limited to,cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl,cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl,cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl, naphthyl,indanyl, adamantyl, and tetrahydronaphthyl. Bridged rings are alsoincluded in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane, and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In one embodiment, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl and tetrahydronaphthyl) and spirorings are also included.

As used herein, the term “glycoside” means any molecule in which a sugargroup is bonded through its anomeric carbon to another group. Examplesof glycosides include, for example methyl α-D-glucopyranoside

methyl β-D-glucopyranoside

glucoside, galactoside, lactoside, lactosidoglycoside, maltoside, etc.Because a glycoside is bonded through its anomeric carbon to anothergroup, it is also known as a non-reducing sugar (i.e., it is not subjectto attack by reagents that attack carbonyl groups).

As used herein, the term “heterocycle” or “heterocyclic” is intended tomean any stable monocyclic, bicyclic, or tricyclic ring which issaturated, unsaturated, or aromatic and comprises carbon atoms and oneor more ring heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, independently selected from the group consisting ofnitrogen, oxygen, and sulfur. A bicyclic or tricyclic heterocycle mayhave one or more heteroatoms located in one ring, or the heteroatoms maybe located in more than one ring. The nitrogen and sulfur heteroatomsmay optionally be oxidized (i.e., N→O and S(O)_(p), where p=1 or 2).When a nitrogen atom is included in the ring it is either N or NH,depending on whether or not it is attached to a double bond in the ring(i.e., a hydrogen is present if needed to maintain the tri-valency ofthe nitrogen atom). The nitrogen atom may be substituted orunsubstituted (i.e., N or NR wherein R is H or another substituent, asdefined). The heterocyclic ring may be attached to its pendant group atany heteroatom or carbon atom that results in a stable structure. Theheterocyclic rings described herein may be substituted on carbon or on anitrogen atom if the resulting compound is stable. A nitrogen in theheterocycle may optionally be quaternized. In one embodiment, when thetotal number of S and O atoms in the heterocycle exceeds 1, then theseheteroatoms are not adjacent to one another. Bridged rings are alsoincluded in the definition of heterocycle. A bridged ring occurs whenone or more atoms (i.e., C, O, N, or S) link two non-adjacent carbon ornitrogen atoms. Bridges include, but are not limited to, one carbonatom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and acarbon-nitrogen group. It is noted that a bridge always converts amonocyclic ring into a tricyclic ring. When a ring is bridged, thesubstituents recited for the ring may also be present on the bridge.Spiro and fused rings are also included.

As used herein, the term “aromatic heterocycle” or “heteroaryl” isintended to mean a stable 5, 6, or 7-membered monocyclic or bicyclicaromatic heterocyclic ring or 7, 8, 9, 10, 11, or 12-membered bicyclicaromatic heterocyclic ring which consists of carbon atoms and one ormore heteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6heteroatoms, independently selected from the group consisting ofnitrogen, oxygen, and sulfur. In the case of bicyclic heterocyclicaromatic rings, only one of the two rings needs to be aromatic (e.g.,2,3-dihydroindole), though both may be (e.g., quinoline). The secondring can also be fused or bridged as defined above for heterocycles. Thenitrogen atom may be substituted or unsubstituted (i.e., N or NR whereinR is H or another substituent, as defined). The nitrogen and sulfurheteroatoms may optionally be oxidized (i.e., N→O and S(O)_(p), wherep=1 or 2). It is to be noted that total number of S and O atoms in thearomatic heterocycle is not more than 1.

Examples of heterocycles include, but are not limited to, acridinyl,azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5 (4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.

“Acyl” includes compounds and moieties that contain the acyl radical(CH₃CO—) or a carbonyl group. “Substituted acyl” includes acyl groupswhere one or more of the hydrogen atoms are replaced by for example,alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl,alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl,phosphate, phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Acylamino” includes moieties wherein an acyl moiety is bonded to anamino group. For example, the term includes alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido groups.

“Aroyl” includes compounds and moieties with an aryl or heteroaromaticmoiety bound to a carbonyl group. Examples of aroyl groups includephenylcarboxy, naphthyl carboxy, etc.

“Alkoxyalkyl”, “alkylaminoalkyl” and “thioalkoxyalkyl” include alkylgroups, as described above, which further include oxygen, nitrogen orsulfur atoms replacing one or more hydrocarbon backbone carbon atoms,e.g., oxygen, nitrogen or sulfur atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups (or alkoxyl radicals) include methoxy, ethoxy,isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples ofsubstituted alkoxy groups include halogenated alkoxy groups. The alkoxygroups can be substituted with groups such as alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano,amino (including alkylamino, dialkylamino, arylamino, diarylamino, andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.Examples of halogen substituted alkoxy groups include, but are notlimited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,chloromethoxy, dichloromethoxy, and trichloromethoxy.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two different carbon atoms or heteroatoms.For example, the term includes “alkoxyalkyl” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to another alkyl group.

The term “ester” includes compounds and moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc. The alkyl, alkenyl, or alkynyl groups are asdefined above.

The term “thioether” includes compounds and moieties which contain asulfur atom bonded to two different carbon or heteroatoms. Examples ofthioethers include, but are not limited to alkthioalkyls,alkthioalkenyls, and alkthioalkynyls. The term “alkthioalkyls” includecompounds with an alkyl, alkenyl, or alkynyl group bonded to a sulfuratom which is bonded to an alkyl group. Similarly, the term“alkthioalkenyls” and alkthioalkynyls” refer to compounds or moietieswherein an alkyl, alkenyl, or alkynyl group is bonded to a sulfur atomwhich is covalently bonded to an alkynyl group.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

“Polycyclyl” or “polycyclic radical” refers to two or more cyclic rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings. Rings that are joined through non-adjacent atoms are termed“bridged” rings. Each of the rings of the polycycle can be substitutedwith such substituents as described above, as for example, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato,phosphinato, cyano, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

An “anionic group,” as used herein, refers to a group that is negativelycharged at physiological pH. Anionic groups include carboxylate,sulfate, sulfonate, sulfinate, sulfamate, tetrazolyl, phosphate,phosphonate, phosphinate, or phosphorothioate or functional equivalentsthereof “Functional equivalents” of anionic groups are intended toinclude bioisosteres, e.g., bioisosteres of a carboxylate group.Bioisosteres encompass both classical bioisosteric equivalents andnon-classical bioisosteric equivalents. Classical and non-classicalbioisosteres are known in the art (see, e.g., Silverman, R. B. TheOrganic Chemistry of Drug Design and Drug Action, Academic Press, Inc.:San Diego, Calif., 1992, pp. 19-23). In one embodiment, an anionic groupis a carboxylate.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers such as geometrical isomer,optical isomer based on an asymmetrical carbon, stereoisomer, tautomerand the like which occur structurally and an isomer mixture and is notlimited to the description of the formula for convenience, and may beany one of isomer or a mixture. Therefore, an asymmetrical carbon atommay be present in the molecule and an optically active compound and aracemic compound may be present in the present compound, but the presentinvention is not limited to them and includes any one. In addition, acrystal polymorphism may be present but is not limiting, but any crystalform may be single or a crystal form mixture, or an anhydride orhydrate. Further, so-called metabolite which is produced by degradationof the present compound in vivo is included in the scope of the presentinvention.

“Isomerism” means compounds that have identical molecular formulae butthat differ in the nature or the sequence of bonding of their atoms orin the arrangement of their atoms in space. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers”.Stereoisomers that are not mirror images of one another are termed“diastereoisomers”, and stereoisomers that are non-superimposable mirrorimages are termed “enantiomers”, or sometimes optical isomers. A carbonatom bonded to four nonidentical substituents is termed a “chiralcenter”.

“Chiral isomer” means a compound with at least one chiral center. It hastwo enantiomeric forms of opposite chirality and may exist either as anindividual enantiomer or as a mixture of enantiomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture”. A compound that has more thanone chiral center has 2^(n-1)-enantiomeric pairs, where n is the numberof chiral centers. Compounds with more than one chiral center may existas either an individual diastereomer or as a mixture of diastereomers,termed a “diastereomeric mixture”. When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn etal., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem.Educ. 1964, 41, 116).

“Geometric Isomers” means the diastereomers that owe their existence tohindered rotation about double bonds. These configurations aredifferentiated in their names by the prefixes cis and trans, or Z and E,which indicate that the groups are on the same or opposite side of thedouble bond in the molecule according to the Cahn-Ingold-Prelog rules.

Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

Additionally, the compounds of the present invention, for example, thesalts of the compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

“Tautomers” refers to compounds whose structures differ markedly inarrangement of atoms, but which exist in easy and rapid equilibrium. Itis to be understood that the compounds of the invention may be depictedas different tautomers. It should also be understood that when compoundshave tautomeric forms, all tautomeric forms are intended to be withinthe scope of the invention, and the naming of the compounds does notexclude any tautomer form.

Some compounds of the present invention can exist in a tautomeric formwhich are also intended to be encompassed within the scope of thepresent invention.

The compounds, salts and prodrugs of the present invention can exist inseveral tautomeric forms, including the enol and imine form, and theketo and enamine form and geometric isomers and mixtures thereof. Allsuch tautomeric forms are included within the scope of the presentinvention. Tautomers exist as mixtures of a tautomeric set in solution.In solid form, usually one tautomer predominates. Even though onetautomer may be described, the present invention includes all tautomersof the present compounds.

A tautomer is one of two or more structural isomers that exist inequilibrium and are readily converted from one isomeric form to another.This reaction results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent, and pH. The conceptof tautomers that are interconvertable by tautomerizations is calledtautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism, is exhibited by glucose.It arises as a result of the aldehyde group (—CHO) in a sugar chainmolecule reacting with one of the hydroxy groups (—OH) in the samemolecule to give it a cyclic (ring-shaped) form.

Tautomerizations are catalyzed by: Base: 1. deprotonation; 2. formationof a delocalized anion (e.g. an enolate); 3. protonation at a differentposition of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g. in thenucleobases guanine, thymine, and cytosine), amine-enamine andenamine-enamine. Examples include:

It will be noted that the structure of some of the compounds of theinvention include asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of theinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Alkenes can include either the E- or Z-geometry,where appropriate. The compounds of this invention may exist instereoisomeric form, therefore can be produced as individualstereoisomers or as mixtures.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative”, refers to compounds that havea common core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byformula I are indole derivatives, and have formula I as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres includeacyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

A “pharmaceutical composition” is a formulation containing the disclosedcompounds in a form suitable for administration to a subject. In oneembodiment, the pharmaceutical composition is in bulk or in unit dosageform. The unit dosage form is any of a variety of forms, including, forexample, a capsule, an IV bag, a tablet, a single pump on an aerosolinhaler, or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate, orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

A “subject” includes mammals, e.g., humans, companion animals (e.g.,dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs,horses, fowl, and the like) and laboratory animals (e.g., rats, mice,guinea pigs, birds, and the like). In one embodiment, the subject ishuman.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

The compounds of the invention are capable of further forming salts. Allof these forms are also contemplated within the scope of the claimedinvention.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines, alkali or organic salts ofacidic residues such as carboxylic acids, and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include, but are not limited to, thosederived from inorganic and organic acids selected from 2-acetoxybenzoic,2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic,bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic,lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic,phosphoric, polygalacturonic, propionic, salicyclic, stearic, subacetic,succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluenesulfonic, and the commonly occurring amine acids, e.g., glycine,alanine, phenylalanine, arginine, etc.

Other examples include hexanoic acid, cyclopentane propionic acid,pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamicacid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, andthe like. The invention also encompasses salts formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base such as ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile can be used.Lists of suitable salts are found in Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990). For example, saltscan include, but are not limited to, the hydrochloride and acetate saltsof the aliphatic amine-containing, hydroxyl amine-containing, andimine-containing compounds of the present invention.

The compounds of the present invention can also be prepared as esters,for example pharmaceutically acceptable esters. For example a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group ina compound can be converted to its corresponding ester, e.g., anacetate, propionate, or other ester.

The compounds of the present invention can also be prepared as prodrugs,for example pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxy, amino,sulfhydryl, carboxy, or carbonyl group is bonded to any group that, maybe cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters groups (e.g. ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g. N-acetyl) N-Mannich bases, Schiff bases and enaminonesof amino functional groups, oximes, acetals, ketals and enol esters ofketone and aldehyde functional groups in compounds of Formula I, and thelike, See Bundegaard, H. “Design of Prodrugs” pl-92, Elesevier, NewYork-Oxford (1985).

“Protecting group” refers to a grouping of atoms that when attached to areactive group in a molecule masks, reduces or prevents that reactivity.Examples of protecting groups can be found in Green and Wuts, ProtectiveGroups in Organic Chemistry, (Wiley, 2^(nd) ed. 1991); Harrison andHarrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8(John Wiley and Sons, 1971-1996); and Kocienski, Protecting Groups,(Verlag, 3^(rd) ed. 2003).

The term “amine protecting group” is intended to mean a functional groupthat converts an amine, amide, or other nitrogen-containing moiety intoa different chemical group that is substantially inert to the conditionsof a particular chemical reaction. Amine protecting groups arepreferably removed easily and selectively in good yield under conditionsthat do not affect other functional groups of the molecule. Examples ofamine protecting groups include, but are not limited to, formyl, acetyl,benzyl, t-butyldimethylsilyl, t-butdyldiphenylsilyl, t-butyloxycarbonyl(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,2-trimethylsilyl-ethyoxycarbonyl, 1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl(NVOC), and the like. Other suitable amine protecting groups arestraightforwardly identified by those of skill in the art.

Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated such as benzyl, and trityl ethersas well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

In the specification, the singular forms also include the plural, unlessthe context clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. In the case of conflict, the present specificationwill control.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

“Combination therapy” (or “co-therapy”) includes the administration of acompound of the invention and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents. The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. The sequence in which the therapeutic agentsare administered is not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where processes are described as having,including, or comprising specific process steps, the processes alsoconsist essentially of, or consist of, the recited processing steps.Further, it should be understood that the order of steps or order forperforming certain actions are immaterial so long as the inventionremains operable. Moreover, two or more steps or actions may beconducted simultaneously.

The compounds, or pharmaceutically acceptable salts thereof, isadministered orally, nasally, transdermally, pulmonary, inhalationally,buccally, sublingually, intraperintoneally, subcutaneously,intramuscularly, intravenously, rectally, intrapleurally, intrathecallyand parenterally. In one embodiment, the compound is administeredorally. One skilled in the art will recognize the advantages of certainroutes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

In one embodiment, the compound is prepared for oral administration,wherein the disclosed compounds or salts thereof are combined with asuitable solid or liquid carrier or diluent to form capsules, tablets,pills, powders, syrups, solutions, suspensions and the like.

The tablets, pills, capsules, and the like contain from about 1 to about99 weight percent of the active ingredient and a binder such as gumtragacanth, acacias, corn starch or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch or alginic acid; a lubricant such as magnesium stearate; and/or asweetening agent such as sucrose, lactose, saccharin, xylitol, and thelike. When a dosage unit form is a capsule, it often contains, inaddition to materials of the above type, a liquid carrier such as afatty oil.

In some embodiments, various other materials are present as coatings orto modify the physical form of the dosage unit. For instance, in someembodiments, tablets are coated with shellac, sugar or both. In someembodiments, a syrup or elixir contains, in addition to the activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavoring such as cherry or orange flavor,and the like.

For some embodiments relating to parental administration, the disclosedcompounds, or salts, solvates, tautomers or polymorphs thereof, can becombined with sterile aqueous or organic media to form injectablesolutions or suspensions. In one embodiment, injectable compositions areaqueous isotonic solutions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. In addition, they may also containother therapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1 to 75%, in another embodiment, thecompositions contain about 1 to 50%, of the active ingredient.

For example, injectable solutions are produced using solvents such assesame or peanut oil or aqueous propylene glycol, as well as aqueoussolutions of water-soluble pharmaceutically-acceptable salts of thecompounds. In some embodiments, dispersions are prepared in glycerol,liquid polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. The terms “parenteraladministration” and “administered parenterally” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal andintrasternal injection and infusion.

For rectal administration, suitable pharmaceutical compositions are, forexample, topical preparations, suppositories or enemas. Suppositoriesare advantageously prepared from fatty emulsions or suspensions. Thecompositions may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.1 to 75%, in another embodiment, compositions contain about 1 to 50%,of the active ingredient.

In some embodiments, the compounds are formulated to deliver the activeagent by pulmonary administration, e.g., administration of an aerosolformulation containing the active agent from, for example, a manual pumpspray, nebulizer or pressurized metered-dose inhaler. In someembodiments, suitable formulations of this type also include otheragents, such as antistatic agents, to maintain the disclosed compoundsas effective aerosols.

A drug delivery device for delivering aerosols comprises a suitableaerosol canister with a metering valve containing a pharmaceuticalaerosol formulation as described and an actuator housing adapted to holdthe canister and allow for drug delivery. The canister in the drugdelivery device has a headspace representing greater than about 15% ofthe total volume of the canister. Often, the polymer intended forpulmonary administration is dissolved, suspended or emulsified in amixture of a solvent, surfactant and propellant. The mixture ismaintained under pressure in a canister that has been sealed with ametering valve.

For nasal administration, either a solid or a liquid carrier can beused. The solid carrier includes a coarse powder having particle size inthe range of, for example, from about 20 to about 500 microns and suchformulation is administered by rapid inhalation through the nasalpassages. In some embodiments where the liquid carrier is used, theformulation is administered as a nasal spray or drops and includes oilor aqueous solutions of the active ingredients.

Also contemplated are formulations that are rapidly dispersing dosageforms, also known as “flash dose” forms. In particular, some embodimentsof the present invention are formulated as compositions that releasetheir active ingredients within a short period of time, e.g., typicallyless than about five minutes, in another embodiment, less than aboutninety seconds, in another embodiment, less than about thirty secondsand in another embodiment, in less than about ten or fifteen seconds.Such formulations are suitable for administration to a subject via avariety of routes, for example by insertion into a body cavity orapplication to a moist body surface or open wound.

Typically, a “flash dosage” is a solid dosage form that is administeredorally, which rapidly disperses in the mouth, and hence does not requiregreat effort in swallowing and allows the compound to be rapidlyingested or absorbed through the oral mucosal membranes. In someembodiments, suitable rapidly dispersing dosage forms are also used inother applications, including the treatment of wounds and other bodilyinsults and diseased states in which release of the medicament byexternally supplied moisture is not possible.

“Flash dose” forms are known in the art; see for example, effervescentdosage forms and quick release coatings of insoluble microparticles inU.S. Pat. Nos. 5,578,322 and 5,607,697; freeze dried foams and liquidsin U.S. Pat. Nos. 4,642,903 and 5,631,023; melt spinning of dosage formsin U.S. Pat. Nos. 4,855,326, 5,380,473 and 5,518,730; solid, free-formfabrication in U.S. Pat. No. 6,471,992; saccharide-based carrier matrixand a liquid binder in U.S. Pat. Nos. 5,587,172, 5,616,344, 6,277,406,and 5,622,719; and other forms known to the art.

The compounds of the invention are also formulated as “pulsed release”formulations, in which the compound is released from the pharmaceuticalcompositions in a series of releases (i.e., pulses). The compounds arealso formulated as “sustained release” formulations in which thecompound is continuously released from the pharmaceutical compositionover a prolonged period.

Also contemplated are formulations, e.g., liquid formulations, includingcyclic or acyclic encapsulating or solvating agents, e.g.,cyclodextrins, polyethers, or polysaccharides (e.g., methylcellulose),or in another embodiment, polyanionic 3-cyclodextrin derivatives with asodium sulfonate salt group separate from the lipophilic cavity by analkyl ether spacer group or polysaccharides. In one embodiment, theagent is methylcellulose. In another embodiment, the agent is apolyanionic β-cyclodextrin derivative with a sodium sulfonate saltseparated from the lipophilic cavity by a butyl ether spacer group,e.g., CAPTISOL® (CyDex, Overland, KS). One skilled in the art canevaluate suitable agent/disclosed compound formulation ratios bypreparing a solution of the agent in water, e.g., a 40% by weightsolution; preparing serial dilutions, e.g. to make solutions of 20%, 10,5%, 2.5%, 0% (control), and the like; adding an excess (compared to theamount that can be solubilized by the agent) of the disclosed compound;mixing under appropriate conditions, e.g., heating, agitation,sonication, and the like; centrifuging or filtering the resultingmixtures to obtain clear solutions; and analyzing the solutions forconcentration of the disclosed compound.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1: Syntheses

Representative syntheses of compounds of the invention are describedherein.

Synthesis of Compounds 1 and 2 (KX1-136 and KX1-305)3-benzyloxybenzonitrile

To a solution of 3-cyanophenol (5.00 g, 42.00 mmol) in acetone (100 ml),potassium carbonate (5.79 g, 42.0 mmol), potassium iodide (335 mg, 21.0mmol) and benzyl bromide (4.20 ml, 42.00 mmol) were added and thereaction mixture refluxed for 12 hrs (TLC, ethyl acetate:hexanel: 1,R_(f)=0.6), then the solvent removed under vacuum and the residueportioned between water (50 ml) and ethyl acetate (50 ml), the organiclayer was washed with water twice and dried over anhydrous sodiumsulfate and evaporated under reduced pressure to give the target etheras a yellow oil (8.46 g) 96% yield; ¹H NMR (DMSO (dimethylsulfoxide),400 MHz): δ 7.51-7.33 (m, 9H), 5.16 (s, 2H).

3-benzyloxybenzylaminehydrochloride

To a suspension of lithium aluminum hydride, LAH (4.314 g, 113.684 mmol)in dry ether (200 ml) a solution of the 3-benzyloxybenzonitrile in ether(7.92 g, 37.894 mmol) was added drop-wise during 10 min at roomtemperature, and allowed to stir for 4 hrs (TLC, ethyl acetate: hexane1:3, R_(f)=0.5), the reaction was quenched with 10 ml ethyl acetate and10 ml water and filtered. The organic layer was washed with water, driedover Na₂SO₄ and treated with 10 ml conc. HCl to form instant whiteprecipitate (6 g) 68% yield. ¹H NMR (DMSO, 400 MHz): δ 8.33 (s, 3H),7.45-7.37 (m, 4H), 7.34-7.30 (m, 2H), 7.19 (s, 1H), 7.02 (t, J=10 Hz,2H), 5.10 (s, 2H), 3.97 (s, 2H).

N(3-benzyloxy-benzyl)-4-biphenylacetamide

To a solution of 4-biphenyl acetic acid (2.29 g, 10.45 mmol) indimethylformamide, DMF, (30 ml) was added diisopropylethylamine, DIEA,(5.47 ml, 31.35 mmol) and stirred at room temperature for 15 min, thenbenzotriazolyloxy-tris[pyrrolidino]-phosphonium hexafluorophosphate,PyBOP™, (5.43 g, 10.45 mmol) was added and the stirring was continuedfor further 30 min, then 3-benzyloxybenzylaminehydrochloride (2.6 g,10.45 mmol) was added and the stirring continued for 24 hrs. Thereaction mixture was then poured on to ice cooled water acidified with(10 ml) 1 N HCl and extracted with ethyl acetate (100 ml) and theorganic layer washed with saturated solution of NaHCO₃, water and brine,dried over Na₂SO₄ and the solvent removed under vacuum to give ayellowish-white powder of the desired compound (2.65 g) 62% yield.

Another procedure involves use amide formation using the acid chlorideas shown in the following reaction.

To 4-biphenylacetic acid (2.5 g) in a flask, thionylchloride (20 ml) wasadded and heated to reflux for 1 h, cooled, and the excessthionylchloride removed under vacuum to dryness, then the produced crudeacid chloride 2.8 g, dissolved in dry DCM (dichloromethane) (30 ml), andadded drop wise at 0° C. to equimolar amount of the3-benzyloxybenzylamine solution in DCM (10 ml) with (1.5 mol) oftriethylamine (TEA) and stirred for 5 hrs, then poured onto acidifiedcold water, the organic layer washed with water, brine and the solventremoved under reduced pressure to give the target amide in 80% yield. ¹HNMR (DMSO, 500 MHz): δ 8.58 (t, J=12 Hz 1H), 7.60-7.57 (m, 4H),7.44-7.29 (m, 10H), δ 7.21 (t, J=16.5 Hz, 2H), 6.85 (d, J=6.5 Hz, 2H),6.81 (d, J=8.0 Hz, 1H), 5.00 (s, 2H), 4.24 (d, J=6 Hz, 2H), 3.51 (s,2H).

Compound 1: N(3-hydroxy-benzyl)-4-biphenylacetamide

To remove the benzyl group of this ether (5.00 g, 13.35 mmol) wasdissolved in methanol (20 ml), to this solution was added a catalyticamount of 10% Pd/C (355 mg, 2.21 mmol) in a Parr hydrogenator (55 psi)for 5 hrs, filtered through celite and the solvent removed under vacuumto give the target phenol as yellowish powder (3.20 g) 84% yield, whichcrystallized from methanol to give (1.5 g) of white crystallinematerial, mp=169-170° C. ¹H NMR (DMSO, 400 MHz): δ 9.34 (s, 1H), 8.53(s, 1H), 7.63 (d, J=8 Hz, 2H), 7.58 (d, J=8.4 Hz, 2H), 7.44 (t, J=7.6Hz, 2H), 7.35 (d, J=8 Hz, 3H), 7.07 (t, J=8 Hz, 1H), 6.65-6.60 (m, 3H),4.17 (d, J=5.6 Hz, 2H), 3.5 (s, 2H). FAB (fast atom bombardment) HRMSm/e calcd. For (M+H) C₂₁H₂₀NO₂: 318.1449; found: 318.1484.

Compound 2: N(3-fluoro-benzyl)-4-biphenylacetamide

To a solution of 4-biphenyl acetic acid (2.00 g, 9.42 mmol) in DMF (20ml) was added DIEA (3.29 ml, 18.84 mmol) and stirred at room temperaturefor 15 min, then PyBOP (4.90 g, 9.42 mmol) added and the stirringcontinued for further 30 min, then 3-fluorobenzylamine (1.18 g, 9.42mmol) added and the stirring continued for 24 hrs, then the reactionmixture poured on to ice cooled water acidified with (10 ml) 1 N HCl andextracted with ethyl acetate (100 ml) and the organic layer washed withsaturated solution of NaHCO₃, water and brine, dried over Na₂SO₄ and thesolvent removed under vacuum to give a white powder of the desiredcompound (1.00 g) 33% yield. Another method involves the acid chloridecoupling method described below.

4-biphenylacetic acid (2.5 g, 11.78 mmol) charged in a flask thenthionylchloride (15 ml) was added and heated to reflux for 1 h, cooled,and the excess thionylchloride removed under vacuum to dryness, then theproduced crude acid chloride (2.8 g, 12.13 mmol) dissolved in dry DCM(30 ml), and added drop wise at 0° C. to (1.38 ml, 12.13 mmol) of the3-fluorobenzylamine solution in DCM (10 ml) along with (1.69 ml, 12.13mmol) of TEA and stirred for 5 hrs, then poured onto acidified coldwater, the organic layer washed with water, brine and the solventremoved under reduced pressure to give the target amide (3.1 g) 80%yield. Recrystallized from methanol, mp=170-172° C. ¹H NMR (DMSO, 500MHz): δ 8.62 (t, J=11 Hz, 1H), 7.63 (d, J=8 Hz, 2H), 7.59 (d, J=8.5 Hz,2H), 7.44 (t, J=7.5 Hz, 2H), 7.37-7.31 (m, 4H), 7.08-7.01 (m, 3H), 4.28(d, J=5.5 Hz, 2H), 3.52 (s, 2H). FAB HRMS m/e calcd. For (M+H)C₂₁H₁₈FNO: 320.1406; found: 320.2, and the base peak found: 342.1262 for(M+Na); calcd. 342.1372.

Synthesis of Compound 3, KX1-306

The synthesis, outlined in Scheme 1, began with acid chloride formationof biphenylacetic acid followed by amide coupling with3,5-dibenzyloxybenzylamine. A large number of impurities were introducedby acid chloride formation. However, other amide coupling proceduressuch as, for example, PyBOP or carbodiimides, can also be used in thisreaction.

Cleavage of one of the benzyl groups was accomplished under highpressure hydrogen (50-60 psi) for 15 hours. The reaction was monitoredby TLC. Silica gel chromatography was used to separate the product fromthe starting material as well as the dihydroxy side-product.

Biphenyl acetic acid (220 mg, 1.00 mmol) was dissolved in DCM, 5 eq(0.38 mL) of thionyl chloride were added and the reaction was refluxedfor 4 hours. Solvents were removed in vacuo and the residue wasdissolved in DCM. 3,5-Dibenzyloxybenzylamine (1.1 eq) was added followedby TEA (1 eq). The reaction was then stirred at room temperatureovernight. The reaction was diluted to 45 mL (with DCM) and washed with1 N HCl (3×20 L), saturated sodium bicarbonate (3×20 mL), and brine(3×20 mL). The Reaction was then dried with sodium sulfate and removedin vacuo to give 330 mg of crude product. Silica gel chromatography (1:1DCM:EtOAc (ethyl acetate)) gave 220 mg pure product. TLC Rf=0.2 (singlespot, 7:3 hexanes:EtOAc). LCMS 514.2 (m+H) 536.2 (m+Na). ¹H NMR (300MHz, CDCl₃) δ (ppm) 3.65 (s, 2H), 4.50 (d, 5.7 Hz, 2H), 4.96 (s, 4H),5.71 (s, 1H), 6.43 (s, 2H), 6.49 (s, 1H), 7.58-7.26 (m, 19H).

The dibenzyloxyamide (1) was dissolved in 15 ml EtOAc (ethyl acetate)with gentle heating in a Parr bottle. This was put on the hydrogenatorat 50 psi hydrogen for 15 hr. The reaction was filtered through celiteand the solvent was removed in vacuo to give a crude mixture of startingmaterial and product. Silica gel chromatography gave 50 mg 1 and 41 mgdesired product KX1-306; LCMS 424.1 (m+H), 446.2 (m+Na), 847.0 (2m+H),868.9 (2m+Na). ¹H NMR (400 MHz, CDCl₃) δ (ppm) 3.66 (s, 2H), 4.38 (d,5.6 Hz, 2H), 4.98 (s, 2H), 5.71 (s, 1H), 6.43 (s, 2H), 6.49 (s, 1H),7.30-7.45 (m, 10H), 7.54-7.57 (m, 4H).

Synthesis of Compound 4, KX1-307

The synthesis is outlined in Scheme 2. In one synthesis, the reactioncommenced with amide bond formation to give 2, followed by a Suzukicoupling with phenylboronic acid to give the meta-biphenyl productCompound 4, KX1-307. In the Suzuki reaction, the biphenyl product wasformed but the reaction did not go to completion (by NMR and LCMS)despite additional, time, heat, and extra catalyst. Using silica gelchromatography, the product could not be separated from the bromostarting material 2. Reversing the Suzuki and amide coupling solved theseparation problem and successfully produced the metabiphenyl amideKX1-307 as well as 2′-Fluorobiphenyl-4-acetamide KX1-309 (compound 6,Scheme 3).

3-Bromophenylacetic acid (250 mg, 1.163 mmol) and 156 mg (1.1 eq) ofphenylboronic acid were dissolved in 6 mL water:isopropanol (6:1).Sodium carbonate (160 mg, 1.3 eq) was dissolved in 0.5 mL distilledwater and added to the reaction followed by Pd(OH)₂/C (74 mg, 3 mol %).This was rotated in a 65° C. water bath for 5 hours. The reaction wasfiltered through filter paper. Filter paper was washed with 25 mLisopropanol:water:1 N NaOH (35:5:1). Washes were combined and acidifiedto pH 2 with 1 N sulfuric acid. Isopropanol was removed in vacuo andwater (10 mL) was added. This aqueous layer was washed withdichloromethane (3×20 mL). Organic washes were combined, dried withsodium sulfate, and removed in vacuo to give 215 mg (87% yield) of thebiphenyl product 3. TLC Rf=0.7 (long streak, 1:1 EtOAc:DCM). ¹H NMR (300MHz, CDCl₃) δ (ppm) 3.72 (s, 2H), 7.26-7.60 (m, 9H).

3-Biphenylacetic acid (3) (100 mg, 0.472 mmol), 3-Fluorobenzylamine (1.1eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDCI,(1.1 eq), and HOBT (1-hydroxyenzotriazole, 1.0 eq) were all dissolved in10 mL anhydrous DCM. After 10 min DIEA (1.1 eq) was added and thereaction was allowed to go overnight. The reaction was diluted to 25 mLand washed with 1N HCl (3×10 L), saturated sodium bicarbonate (3×10 mL),and brine (2×20 mL). The reaction was dried with sodium sulfate andremoved in vacuo to give 124 mg pure KX1-307 (83% yield). TLC Rf=0.7(single spot, 1:1 EtOAc:DCM). ¹H NMR (300 MHz, CDCl₃) δ (ppm) 3.69 (s,2H) 4.40 (d, 6.0 Hz) 5.77 (s, 1H) 6.86-6.96 (m, 3H) 7.10-7.26 (m, 2H)7.32 (m, 8H).

Synthesis of Compound 6, KX-309

The synthesis is outlined in Scheme 3. 4-Bromophenylacetic acid (500 mg,2.33 mmol) and 358 mg of 2-fluorophenylboronic acid (1.1 eq) weredissolved in 12 mL, 6:1 water:isopropanol. Sodium carbonate (320 mg, 1.3eq) was dissolved in 1 mL distilled water and added to the reactionfollowed by Pd(OH)₂/C (148 mg, 3 mol %). This was rotated in a 65° C.water bath for 5 hours. The reaction was filtered through filter paper.Filter paper was washed with 50 mL isopropanol:water:1 N NaOH (35:5:1).Washes were combined and acidified to pH 2 with 1 N sulfuric acid.Isopropanol was removed in vacuo, water (20 mL) was added and washedwith dichloromethane (3×30 mL). Organic washes were combined, dried withsodium sulfate, and removed in vacuo to give 177 mg (35% yield) of thebiphenyl product 4. TLC Rf=0.7 (long streak, 1:1 EtOAc:DCM). ¹H NMR (500MHz, CDCl₃) δ (ppm) 3.73 (s, 2H), 7.16 (t, 10.5 Hz, 1H), 7.22 (t, 7.5Hz, 1H), 7.32 (qd, 1.5 Hz, 7.5 Hz, 1H), 7.38 (d, 8.0 Hz, 2H), 7.44 (td,1.5 Hz, 7.5 Hz, 1H), 7.54 (d, 8.0 Hz, 2H).

2′-Fluorobiphenylacetic acid (4) (103 mg, 0.448 mmol),3-Fluorobenzylamine (1.1 eq), EDCI (1.1 eq), and HOBT (1.0 eq) were alldissolved in 6 mL anhydrous DCM. After 10 min DIEA (1.1 eq) was addedand the reaction was allowed to go overnight. Reaction was diluted to 25mL and washed with 1 N HCl (3×10 L), saturated sodium bicarbonate (3×10mL), and brine (2×20 mL). The reaction was dried with sodium sulfate andremoved in vacuo to give 126 mg pure Compound 6, KX1-309 (83% yield).LCMS 360.1 (m+Na) 696.8 (2m+Na). ¹H NMR (300 MHz, CDCl₃) δ (ppm) 3.67(s, 2H) 4.21 (d, 6.0 Hz, 2H) 5.79 (s, 1H) 6.87-6.98 (m, 3H) 7.10-7.44(m, 7H) 7.53 (dd, 1.5 Hz, 7.5 Hz, 2H).

Synthesis of Compound 5: N-(3-fluorophenyl)-4-biphenylacetamide, KXI-308

Thionyl chloride (0.38 ml, 5.0 mmole) was added to an ice water cooledsolution of 4-Biphenylacetic acid (0.2 g, 0.9 mmole) in 5 mldichloromethane, solution allowed to warm to room temperature thenheated under reflux for 1 hr, the solvent and excess thionyl chloridewas evaporated under vacuum, the oil formed was redissolved in 5 mldichloromethane followed by addition of 4-Dimethylaminopyridine (0.12gm, 1.0 mmole) and 3-Fluoroaniline (0.11 gm, 1.0 mmole), stirred at roomtemperature over night, then the reaction mixture was diluted with 10 mldichloromethane and 20 ml water, the organic layer washed with 1 N HCl,saturated NaHCO₃ solution, and saturated NaCl solution, dried usingNa₂SO₄ and evaporated dryness (0.2 gm, 72%), H¹-NMR INOVA-500 (CDCl₃) δ3.805 (s, 2H), 6.815 (t, J=8.5 Hz, 1H), 7.068 (d, J=8.0 Hz, 1H),7.218-7.284 (m, 2H), 7.380-7.499 (m, 6H) 7.620-7.664 (m, 4H). MS (m/z)306.2 (M+H)⁺.

Synthesis of Compound 7: N-(3-fluorobenzyl)-4-(3-fluorophenyl)phenylacetamide, KX1-310

Synthesis of (4′-Fluoro-biphenyl-4-yl)-acetic acid: 4-Bromo-phenylaceticacid (0.5 gm, 2.3 mmole), 3-fluorophenylboronic acid (0.36 gm, 2.4mmole) and 50% water wet 10% Palladium carbon (0.16 gm, 0.075 mmole Pd)were added to 10 ml of 5:1 water isopropanol mixture, then Na₂CO₃ (0.32gm, 3 mmole) dissolved in 3 ml of water was added to the above mixture,the reaction was heated at 65-70° C. overnight, the reaction was cooledto room temperature, diluted with 20 ml of 70:15:1 i-PrOH/H₂O/10% NaOH,filtered, the catalyst was washed with 20 ml×3 using the above mixture,the filtrate was acidified using 20% H₂SO₄, filtered and dried(3′-Fluoro-biphenyl-4-yl)-acetic acid: (0.4 gm, 75%) H¹-NMR INOVA-500(DMSO d₆) δ 3.623 (s, 2H), 7.192 (m, 1H), 7.358 (d, J=8.0 Hz, 2H),7.474-7.515 (m, 3H), 7.652 (d, J=8.0 Hz, 2H), 12.316 (s, 1H).

3-fluorobenzylamine (0.14 ml, 1.1 mmole), PyBOP (0.57 gm, 1.1 mmole),and DIEA (0.36 ml, 2.2 mmole) was dissolved in DMF stirred overnight,the reaction mixture was then poured into water, solid was collected byfiltration, re-crystallized using water-methanol. (0.22 gm, 76%); H¹-NMRINOVA-500 (DMSO d₆) δ 3.550 (s, 2H), 4.303 (d, J=6.5 Hz, 2H),7.027-7.097 (m, 3H), 7.197 (m, 1H), 7.350 (m, 1H), 7.389 (d, J=8.0 Hz,2H), 7.477-7.518 (m, 3H), 7.657 (d, J=8.0 Hz, 2H), 8.652 (t, J=5.5 Hz,1H). MS (m/z) 338.1 (M+H)⁺.

Synthesis of Compound 8, N-(3-fluorobenzyl)-4-(4-fluorophenyl)phenylacetamide, KX1-311

Synthesis of (4′-Fluoro-biphenyl-4-yl)-acetic acid: 4-Bromo-phenylaceticacid (0.5 gm, 2.3 mmole), 4-fluorophenylboronic acid (0.36 gm, 2.4mmole) and 50% water wet 10% Palladium carbon (0.16 gm, 0.075 mmole Pd)were added to 10 ml of 5:1 water isopropanol mixture, then Na₂CO₃ (0.32gm, 3 mmole) dissolved in 3 ml of water was added to the above mixture,the reaction was heated at 65-70° C. overnight, the reaction was cooledto room temperature, diluted with 20 ml of 70:15:1 i-PrOH/H₂O/10% NaOH,filtered, the catalyst was washed with 20 ml×3 using the above mixture,the filtrate was acidified using 20% H₂SO₄, filtered and dried (0.4 gm,75%) H¹-NMR INOVA-500 (DMSO d₆) δ 3.621 (s, 2H), 7.290 (t, J=8.5 Hz,2H), 7.351 (d, J=7.5 Hz, 2H), 7.593 (d, J=7.5 Hz, 2H), 7.695 (t, J=7 Hz,2H), 12.386 (s, 1H).

(4′-Fluoro-biphenyl-4-yl)-acetic acid (0.2 gm, 0.9 mmole),3-fluorobenzylamine (0.14 ml, 1.1 mmole), PyBOP (0.57 gm, 1.1 mmole),and DIEA (0.36 ml, 2.2 mmole) was dissolved in DMF stirred overnight,the reaction mixture was then poured into water, solid was collected byfiltration, re-crystallized using water-methanol. (0.26 gm, 90%); H¹-NMRINOVA-500 (DMSO d₆) δ 3.541 (s, 2H), 4.304 (d, J=5.5 Hz, 2H),7.027-7.098 (m, 3H), 7.273-7.382 (m, 5H), 7.582 (d, J=8.0, 2H), 7.694(m, 2H), 8.641 (t, J=5.5 Hz, 2H) MS (m/z) 338.1 (M+H)⁺.

Synthesis of Compound 9,N-(3-fluorobenzyl)-N-methyl-4-biphenylacetamide, KX1-312

4-biphenylacetic acid (0.25 gm, 1.2 mmole), N-methyl-3-fluorobenzylamine(0.16 gm, 1.2 mmole), EDCI (0.23 gm, 1.2 mmole), and DIEA (0.42 ml, 2.4mmole) was dissolved in 10 ml DCM and stirred overnight. The reactionmixture was diluted with 10 ml of DCM washed with 10% HCl, saturatedNaHCO₃ solution, and saturated NaCl solution, dried using Na₂SO₄ andevaporated to produce viscous clear oil (160 mg, 43%), H¹-NMR INOVA-500(DMSO d₆) indicated the presence of a mixture of cis and trans isomersin a ratio of 1:2, running the NMR experiment was run at 50° C. slightlychange the value for the chemical shift, but had almost no effect on theratio. Protons are labeled H_(a) or H_(b) to indicate it belongs to oneisomer or the other. H¹-NMR INOVA-500 (DMSO d₆) 2.813 (s, 3H_(a)), 3.000(s, 3H_(b)), 3.784 (s, 2H_(a)), 3.841 (s, 2H_(b)), 4.543 (s, 2H_(b)),4.681 (s, 2H_(a)), 6.931-7.649 (m, 13H_(a)+13 H_(b)). MS (m/z) 334.2(M+H)⁺.

Synthesis of Compound 10.N-(3-fluorobenzyl)-4-phenyl-2-fluorophenylacetamide, KX1-313

Synthesis of 4-Bromo-2-fluoro-phenylacetamide:4-Bromo-2-fluorobenzylbromide (5 gm, 18.7 mmole) was dissolved in 30 mlethanol, to which water solution (10 ml) of KCN (2.43 gm, 37.4 mmole)was added, refluxed overnight, then it was cooled to room temperature,poured into 200 ml of crushed ice, filtered, chromatographed using 1:1ethyl acetate followed by ethyl acetate (the cyano compound washydrolyzed on the silica gel to produce the carboxamide), which wasevaporated to produce white solid, (1.3 gm, 32%) H¹-NMR INOVA-500 (DMSOd₆) δ 3.436 (s, 2H), 7.005 (s, 1H), 7.289 (t, J=8.0 Hz, 1H), 7.361 (d,J=8.0 Hz, 1H), 7.478 (m, 1H), 7.517 (s, 1H).

Synthesis of 4-Bromo-2-fluoro-phenylacetic acid:4-Bromo-2-fluoro-phenylacetamide (1.3 gm) was suspended in 100 ml of 30%NaOH, heating at reflux temperature for 24 hrs, cooled to roomtemperature, washed with DCM and ethyl acetate. The aqueous layer wasacidified with conc. HCl, extracted with ethyl acetate, evaporated; theresidue was crystallized from isopropanol-water to give needle crystals(0.5 gm, 38%) Hi-NMR INOVA-500 (DMSO d₆) δ 3.619 (s, 2H), 7.316 (t,J=8.0 Hz, 1H), 7.379 (dd, J=8.0, 1.5 Hz, 1H), 7.516 (dd, J=8.0, 1.5 Hz,1H), 12.555 (s, 1H).

Synthesis of 4-phenyl-2-fluorophenylacetic acid:4-Bromo-2-fluoro-phenylacetic acid (0.25 gm, 1.1 mmole), phenylboronicacid (0.15 gm, 1.2 mmole) and 50% water wet 10% Palladium carbon (0.07gm, 0.033 mmole Pd) were added to 10 ml of 5:1 water isopropanolmixture, then Na₂CO₃ (0.14 gm, 1.3 mmole) dissolved in 3 ml of water wasadded to the above mixture, the reaction was heated at 65-70° C.overnight, the reaction was cooled to room temperature, diluted with 20ml of 70:15:1 i-PrOH/H₂O/10% NaOH, filtered, the catalyst was washedwith 20 ml×3 using the above mixture, the filtrate was acidified using20% H₂SO₄, filtered and dried (0.2 gm, 83%) H¹-NMR INOVA-500 (DMSO d₆) δ3.675 (s, 2H), 7.382-7.518 (m, 6H), 7.707 (d, J=7.5 Hz, 2H), 12.498 (s,1H).

Synthesis of N-(3-fluorobenzyl)-4-phenyl-2-fluorophenylacetamide:4-phenyl-2-fluorophenylacetic acid (0.2 gm, 0.9 mmole),3-fluorobenzylamine (0.14 ml, 1.1 mmole), PyBOP (0.57 gm, 1.1 mmole),and DIEA (0.36 ml, 2.2 mmole) was dissolved in DMF stirred overnight,the reaction mixture was then poured into water, solid was collected byfiltration, re-crystallized using water-methanol. (0.20 gm, 70%); H¹-NMRINOVA-500 (DMSO d₆) δ 3.612 (s, 2H), 4.318 (d, J=6 Hz, 2H), 7.064-7.117(m, 3H), 7.345-7.503 (m, 7H), 7.695 (d, J=7.5 Hz, 2H), 8.660 (t, J=6 Hz,1H). MS (m/z) 338.1 (M+H)⁺.

Synthesis of Compound 11, N(3-fluorobenzyl)-2-phenylpyridine-5-acetamide, KX1-314

Synthesis of 2-phenylpyridine-5-acetic acid: 2-chloropyridine-5-aceticacid (0.2 gm, 1.21 mmole), phenylboronic acid (0.16 gm, 1.3 mmole) and50% water wet 10% Palladium carbon (0.08 gm, 0.036 mmole Pd) were addedto 10 ml of 5:1 water isopropanol mixture, then Na₂CO₃ (0.15 gm, 1.4mmole) dissolved in 3 ml of water was added to the above mixture, thereaction was heated at 65-70° C. overnight, the reaction was cooled toroom temperature, diluted with 20 ml of 70:15:1 i-PrOH/H₂O/10% NaOH,filtered, the catalyst was washed with 20 ml×3 using the above mixture,the filtrate was dried under vacuum and crude mixture was used withoutany purification in the next step.

Synthesis of N(3-fluorobenzyl)-2-phenylpyridine-5-acetamide: To thecrude from the above reaction, 3-fluorobenzylamine (0.15 gm, 1.2 mmole),PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.32 gm, 2.6 mmole) and wasstirred in DMF overnight. The reaction mixture was then poured intowater; solid was collected by filtration, re-crystallized usingwater-methanol (0.06 gm, 18% in two steps). H¹-NMR INOVA-500 (CDCl₃)δ3.645 (s, 2H), 4.438 (d, J=5.5 Hz, 2H), 5.867 (s, 1H), 6.925-7.009 (m,3H), 7.268 (m, 1H), 7.408-7.493 (m, 3H), 7.735 (m, 2H), 7.965-7.982 (m,2H), 8.582 (s, 1H). MS (m/z) 321.2 (M+H)⁺.

Synthesis of Compound 12,N-(3-Fluoro-benzyl)-2-(4-pyridin-2-yl-phenyl)-acetamide, KX1-315

Synthesis of 4-(2-Pyridinyl)benzylalcohol: 4-(2-Pyridinyl)benzaldehyde(2 gm, 11 mmole), and NaBH₄ (0.42 gm, 11 mmole) were stirred at roomtemperature for 2 hr, ethanol was evaporated, residue dissolved in ethylacetate washed with saturated NaHCO₃ solution, and saturated NaClsolution, dried using Na₂SO₄ and evaporated to produce white solid (1.5gm, 75%).

Synthesis of (4-Pyridin-2-yl-phenyl)-acetic acid: The crude of4-(2-Pyridinyl)benzylalcohol was dissolved in 20 ml DCM, cooled usingice/methanol, triethylamine (1.25 ml, 8.9 mmole) was added followed bymethanesulfonylchloride (0.7 ml, 8.9 mmole) added drop wise over 5minutes. The reaction was allowed to stir at room temperature till theTLC indicated consumption of the starting material (3 hrs), aftercompletion of the reaction, the reaction mixture was washed with water,saturated NaHCO₃ solution, and saturated NaCl solution, dried usingNa₂SO₄ and evaporated to produce yellow oil, the oil produced wasdissolved in 25 ml of 90% ethanol, KCN (1.05 gm, 16.2 mmole) was thenadded and it was heated under reflux overnight. Ethanol was evaporated;solid was washed with 50 ml water and filtered. The solid was dissolvedin 30 ml of conc. HCl, refluxed for 48 hr; charcoal was added refluxedfor 1 hr, filtered. The HCl was evaporated, the solid formed wasdissolved in 5 ml of water, NaOH 1 N was added drop wise whileextracting with ethyl acetate, the ethyl acetate extract was dried withNa₂SO₄ and evaporated to produce white solid (0.6 gm, 35% in 3 steps)H¹-NMR INOVA-500 (DMSO d₆) δ 3.641 (s, 2H), 7.345 (t, J=6.0 Hz, 1H),7.381 (d, J=8.5 Hz, 2H), 7.879 (t, J=8.0 Hz, 1H), 7.951 (d, J=8.0 Hz,1H), 8.034 (d, J=8.0 Hz, 2H), 8.662 (d, J=4.0 Hz, 1H), 12.390 (s, 1H).

Synthesis of N-(3-Fluoro-benzyl)-2-(4-pyridin-2-yl-phenyl)-acetamide:(4-Pyridin-2-yl-phenyl)-acetic acid (0.2 gm, 0.9 mmole),3-fluorobenzylamine (0.14 ml, 1.1 mmole), PyBOP (0.57 gm, 1.1 mmole),and DIEA (0.36 ml, 2.2 mmole) was dissolved in DMF stirred overnight,the reaction mixture was then poured into water, solid was collected byfiltration, re-crystallized using water-methanol. (0.13 gm, 45%); H¹-NMRINOVA-500 (DMSO d₆) δ 3.563 (s, 2H), 4.305 (d, J=6.0 Hz, 2H),7.032-7.095 (m, 3H), 7.332-7.360 (m, 2H), 7.404 (d, J=8.0 Hz, 2H), 7.874(t, J=7.0 Hz, 1H), 7.948 (d, J=8.0 Hz, 1H), 8.034 (d, J=8.0 Hz, 2H),8.659 (d, J=4.Hz, 2H). MS (m/z) 321.2 (M+H)⁺.

Synthesis of Compounds 13 and 24

Syntheses of the pyridyl derivatives, Compound 13, KX1-316, and Compound24, KX1-327, are shown in Scheme 4. The amide was made first with anEDCI coupling to give amide 5. The Suzuki with 3- or 4-pyridylboronicacids was then performed. The basic nature of the pyridine ring wasexploited to purify the product from and remaining starting material.The product was pulled into the aqueous phase away from the startingmaterial using 1 N HCl. After several organic washes the aqueous layerwas basified and the product extracted with ethyl acetate. Thispurification procedure worked well and eliminated the need forchromatography.

KX1-316 (Compound 13)

A flame dried 50 mL round bottom flask with two condensers was chargedwith argon. Dimethoxyethane, 15 mL and 1 mL 2 M potassium carbonate washeated to 45° C. while argon was bubbled through the solution. After 1hour the bromo amide (240 mg, 0.7475 mmol) and 3-pyridylboronic acid (92mg, 1.1 eq) were added. After one hour, Pd(PPh₃)₄ (43 mg, 5 mol %) wasadded neat. Reaction was heated at 65-75° C. for 48 hours. The solventwas poured into a round bottom flask, the remaining residue was washedwith ethyl acetate. Solvents were combined and removed in vacuo. Theresidue was taken up in 20 mL 1 N HCl and washed with ethyl acetate(3×10 mL). The acid layer was then basified with a combination of 2 NNaOH and saturated sodium bicarbonate to pH 8-9. The aqueous layer wasthen washed with ethyl acetate (3×20 mL). Solvent extracts werecombined, dried with sodium sulfate and removed in vacuo. Residue waspurified on silica gel column (1:1 DCM: EtOAc) to give 90 mg of thedesired product (38% yield). TLC, Rf 0.2 (1:1DCM:EtOAc). LCMS 321.3(m+H) 640.8 (2m+Na) 662.9 (2M+Na). ¹H NMR (500 MHz, DMSO) 3.54 (s, 2H)4.29 (d, 6.0 Hz, 2H) 7.00-7.08 (m, 3H) 7.34 (q, 8.0 Hz, 1H) 7.40 (d,10.0 Hz, 2H) 7.47 (dd, 6.0 Hz, 10.0 Hz, 1H) 7.66 (d, 10.0 Hz, 2H) 8.05(dt, 2.5 Hz, 10.0 Hz, 1H) 8.55 (dd, 2.0 Hz, 6.0 Hz, 1H) 6.40 (t, 7.0 Hz,1H) 8.78 (d, 2.5 Hz, 1H).

KX1-327 (Compound 24)

A flame dried 50 mL round bottom flask with two condensers was chargedwith argon. Dimethoxyethane, 15 mL and 1 mL 2 M potassium carbonate washeated to 45° C. while argon was bubbled through the solution. After 1hour the bromo amide (150 mg, 0.4672 mmol) and 4-pyridylboronic acid (57mg, 1 eq) were added. After one hour Pd(PPh₃)₄ (27 mg, 5 mol %) wasadded neat. Reaction was heated at 65-75° C. for 72 hours. The solventwas poured into a round bottom flask, the remaining residue was washedwith ethyl acetate. Solvents were combined and removed in vacuo. Theresidue was taken up in 20 mL 1 N HCl and washed with ethyl acetate(3×10 mL). The acid layer was then basified with a combination of 2 NNaOH and saturated sodium bicarbonate to pH 8-9. The aqueous layer wasthen washed with ethyl acetate (3×20 mL). Solvent extracts werecombined, dried with sodium sulfate and removed in vacuo to give 71 mgof the desired product (48% yield). TLC, Rf 0.2 (1:1DCM:EtOAc). LCMS321.3 (m+H). ¹H NMR (500 MHz, DMSO) 3.56 (s, 2H) 4.29 (d, 6.0 Hz, 2H)7.04 (m, 3H) 7.34 (q, 6.5 Hz, 1H) 7.42 (d, 8.0 Hz, 2H) 7.69 (d, 6.0 Hz,2H) 7.75 (d, 8.5 Hz, 2H) 8.61 (d, 6.0 Hz, 2H) 8.64 (t, 5.5 Hz, 1H).

Synthesis of Compound 14,2-[6-(3-Chloro-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide,KX1-317

Synthesis of 2-(3-Chloro-phenyl)-pyridine-5-acetic acid:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole),3-chlorophenylboronic acid (0.2 gm, 1.3 mmole) and 50% water wet 10%Palladium carbon (0.08 gm, 0.036 mmole Pd) were added to 10 ml of 5:1water isopropanol mixture, then Na₂CO₃ (0.15 gm, 1.4 mmole) dissolved in3 ml of water was added to the above mixture, the reaction was heated at65-70° C. overnight, the reaction was cooled to room temperature,diluted with 20 ml of 70:15:1 i-PrOH/H₂O/10% NaOH, filtered, thecatalyst was washed with 20 ml×3 using the above mixture, the filtratewas dried under vacuum and crude mixture was used without anypurification in the next step.

Synthesis of2-[6-(3-Chloro-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide: Tothe crude from the above reaction, 3-fluorobenzylamine (0.15 gm, 1.2mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.32 gm, 2.6 mmole) andwas stirred in DMF overnight. The reaction mixture was then poured intowater; solid was collected by filtration, re-crystallized usingwater-methanol (0.02 gm, 6% in two steps). H¹-NMR INOVA-500 (DMSO d₆) δ3.611 (s, 2H), 4.314 (d, J=6.0 Hz, 2H), 7.048-7.106 (m, 3H), 7.364 (m,1H), 7.500-7.545 (m, 2H), 7.808 (dd, J=8.0, 2.0 Hz, 1H), 7.997 (d, J=8.0Hz, 1H), 8.046 (d, J=8.0 Hz, 1H), 8.126 (d, J=2.0 Hz, 1H), 8.578 (s,1H), 8.699 (bs, 1H). MS (m/z) 355.2 (M+H)⁺.

Synthesis of Compound 14,2-[6-(4-Ethyl-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide,KX1-318

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol. (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis of2-[6-(4-ethyl-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.125 gm,0.5 mmole), 4-ethylbenzeneboronic acid (0.083 gm, 0.55 mmole) wasdissolved in dimethoxymethane (DME), Na₂CO₃ (0.11 gm, 1 mmole) in 5 mlof water was added to the DME solution, the solution was then degassedfor 30 min (Ar through the solution and vacuum applied for the first 5min), Palladiumtetrakistriphenylphosphine (0.029 gm, 0.025 mmole) wasadded, degassed for additional 15 min, refluxed for 24 hr. The reactionwas allowed to cool to room temperature, filtered, solid washed withethyl acetate; the organic layer was dried, evaporated. The residue waschromatographed using ethyl acetate/hexane 3:2. The product is whitesolid (0.08 gm, 47%). H¹-NMR INOVA-500 (DMSO d₆) δ 1.228 (t, J=7.5 Hz,3H), 2.669 (q, J=7.5 Hz, 2H), 3.590 (s, 2H), 4.321 (d, J=6 Hz, 2H),7.053-7.113 (m, 3H), 7.324-7.375 (m, 3H), 7.766 (dd, J=9.0, 2.0 Hz, 1H),7.887 (d, J=8.5 Hz, 1H), 7.994 (d, J=8.0 Hz, 2H), 8.548 (s, 1H), 8.696(t, J=5.5 Hz, 1H). MS (m/z) 349.3 (M+H)⁺.

Synthesis of Compound 16,N-(3-Fluoro-benzyl)-2-(2-fluoro-biphenyl-4-yl)-acetamide, KX1-319

Synthesis of 2-Fluoro-biphenyl-4-carbaldehyde: 4-Bromo-2-fluoro-biphenyl(2 gm, 8 mmole) was dissolved in 20 ml of anhydrous tetrahydrofuran,THF, cooled to −78° C. under argon (Ar), n-Butyl lithium 2.5 M (3.5 ml,8.8 mmole) was added drop wise over 10 min, and was stirred foradditional 1 hr, DMF anhydrous (0.68 ml, 8.8 mmole) was then added,stirred for additional 1 hr, then warmed to room temperature over 4 hr.It was then quenched with water, extracted with ether, ether was dried,evaporated, the produced compound was purified using 9:1 hexane/ethylacetate, to produce white solid (1 gm, 62.5%); H¹-NMR INOVA-500 (CDCl₃)δ 7.416-7.495 (m, 3H), 7.581-7.661 (m, 4H), 7.723 (d, J=8.0 Hz, 1H),9.991 (s, 1H).

Synthesis of (2-Fluoro-biphenyl-4-yl)-methanol:2-Fluoro-biphenyl-4-carbaldehyde (1 gm, 5 mmole), NaBH₄ were dissolvedin ethanol stirred for 2 hrs, NaOH 10% was added, ethanol wasevaporated, the reaction mixture was extracted with ethyl acetate, theethyl acetate extract was dried with Na₂SO₄ and evaporated to producewhite solid (0.8 gm, 80%). H¹-NMR INOVA-500 (CDCl₃) δ 2.266 (s, 1H),4.683 (s, 2H), 7.142-7.168 (m, 2H), 7.339-7.442 (m, 4H), 7.519-7.535 (m,2H).

Synthesis of (2-Fluoro-biphenyl-4-yl)-acetic acid:(2-Fluoro-biphenyl-4-yl)-methanol (0.75 gm, 3.7 mmole) was dissolved in20 ml DCM, cooled using ice/methanol, triethylamine (0.55 ml, 4.0 mmole)was added followed by methanesulfonylchloride (0.3 ml, 4.0 mmole) addeddrop wise over 5 minutes. The reaction was allowed to stir at roomtemperature till the TLC indicated consumption of the starting material(2 hrs), after completion of the reaction, the reaction mixture waswashed with water, saturated NaHCO₃ solution, and saturated NaClsolution, dried using Na₂SO₄ and evaporated to produce yellow oil, theoil produced was dissolved in 25 ml of 70% ethanol, KCN (0.4 gm, 6mmole) was then added and it was heated under reflux overnight. Ethanolwas evaporated; solid was washed with 50 ml water and filtered. Thesolid was dissolved in 20 ml of ethanol, then 20 ml of conc. H₂SO₄ wasadded, and was refluxed overnight; the solution was allowed to cool toroom temperature, poured to 200 ml of crushed ice, the solid wascollected by vacuum filtration, suspended in 25 ml of NaOH 30%, heatedat reflux temperature for 24 hrs, cooled to room temperature, washedwith DCM and ethyl acetate. The aqueous layer was acidified with conc.HCl, extracted with ethyl acetate, evaporated; the residue wascrystallized from isopropanol-water to give white solid (0.15 gm, 18% in3 steps) H¹-NMR INOVA-500 (DMSO d₆) δ 3.672 (s, 2H), 7.191-7.254 (m,2H), 7.389-7.560 (m, 6H), 12.494 (s, 1H).

Synthesis of N-(3-Fluoro-benzyl)-2-(2-fluoro-biphenyl-4-yl)-acetamide:(2-Fluoro-biphenyl-4-yl)-acetic acid (0.12 gm, 0.5 mmole),3-fluorobenzylamine (0.0.8 ml, 0.6 mmole), PyBOP (0.34 gm, 0.6 mmole),and DIEA (0.22 ml, 1.3 mmole) was dissolved in DMF stirred overnight,the reaction mixture was then poured into water, solid was collected byfiltration, re-crystallized using water-methanol. (0.140 gm, 83%);H¹-NMR INOVA-500 (DMSO d₆) δ 3.580 (s, 2H), 4.316 (d, J=5.5 Hz, 2H),7.037-7.110 (m, 3H), 7.210-7.247 (m, 2H), 7.343-7.372 (m, 2H),7.457-7.501 (m, 3H), 7.544 (d, J=8.0 Hz, 2H), 8.660 (t, J=6.0 Hz, 1H).MS (m/z) 338.1 (M+H)⁺.

Synthesis of Compound 17,N-(3-Fluoro-benzyl)-2-[6-(4-fluoro-phenyl)-pyridin-3-yl]-acetamide,KX1-320

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol. (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis ofN-(3-Fluoro-benzyl)-2-[6-(4-fluoro-phenyl)-pyridin-3-yl]-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.093 gm,0.33 mmole), 4-fluorobenzeneboronic acid (0.052 gm, 0.37 mmole) wasdissolved in DME, Na₂CO₃ (0.07 gm, 0.66 mmole) in 5 ml of water wasadded to the DME solution, the solution was then degassed for 30 min (Arthrough the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.016 gm, 0.015 mmole) was added,degassed for additional 15 min, refluxed for 24 hr. The reaction wasallowed to cool to room temperature, filtered, solid washed with ethylacetate; the organic layer was dried, evaporated. The residue waschromatographed using ethyl acetate/hexane 3:2. then it crystallizedfrom methanol-water to produce white solid (0.013 gm, 12%). H¹-NMRINOVA-500 (DMSO d₆) δ 3.587 (s, 2H), 4.306 (d, J=5.0 Hz, 2H),7.041-7.099 (m, 3H), 7.295-7.363 (m, 3H), 7.777 (d, J=7.5, 1H), 7.913(d, J=8.0 Hz, 1H), 8.119 (s, 2H), 8.546 (s, 1H), 8.702 (s, 1H). MS (m/z)339.2 (M+H)⁺.

Synthesis of Compound 18,N-(3-Fluoro-benzyl)-2-[6-(3-fluoro-phenyl)-pyridin-3-yl]-acetamide,KX1-321

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol. (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis ofN-(3-Fluoro-benzyl)-2-[6-(3-fluoro-phenyl)-pyridin-3-yl]-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.125 gm,0.5 mmole), 3-fluorobenzeneboronic acid (0.08 gm, 0.55 mmole) wasdissolved in DME, Na₂CO₃ (0.11 gm, 1.0 mmole) in 5 ml of water was addedto the DME solution, the solution was then degassed for 30 min (Arthrough the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.029 gm, 0.025 mmole) was added,degassed for additional 15 min, refluxed for 24 hr. The reaction wasallowed to cool to room temperature, filtered, solid washed with ethylacetate; the organic layer was dried, evaporated. The residue waschromatographed using ethyl acetate/hexane 3:2, then it crystallizedfrom methanol-water to produce white solid (0.075 gm, 45%). H¹-NMRINOVA-500 (DMSO d₆) δ 3.614 (s, 2H), 4.318 (d, J=6.0 Hz, 2H),7.053-7.099 (m, 3H), 7.273 (t, J=9.0 Hz, 1H), 7.367 (q, J=7.0 Hz, 1H),7.542 (q, J=7.0 Hz, 1H), 7.812 (d, J=8.0 Hz, 1H), 7.891 (d, J=10.0 Hz,1H), 7.942 (d, J=7.5 Hz, 1H), 7.992 (d, J=8.0 Hz, 1H), 8.583 (s, 1H),8.717 (s, 1H). MS 339.2 (M+H)⁺.

Synthesis of Compound 19,2-[6-(3-Ethoxy-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide,KX1-322

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol. (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis ofN-(3-Fluoro-benzyl)-2-[6-(3-fluoro-phenyl)-pyridin-3-yl]-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.15 gm,0.54 mmole), 3-ethoxybenzeneboronic acid (0.096 gm, 0.6 mmole) wasdissolved in DME, Na₂CO₃ (0.11 gm, 1.08 mmole) in 5 ml of water wasadded to the DME solution, the solution was then degassed for 30 min (Arthrough the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.031 gm, 0.027 mmole) was added,degassed for additional 15 min, refluxed for 24 hr. The reaction wasallowed to cool to room temperature, filtered, solid washed with ethylacetate; the organic layer was dried, evaporated. The residue waschromatographed using ethyl acetate/hexane 3:2. then it crystallizedfrom methanol-water to produce white solid (0.03 gm, 17%). H¹-NMRINOVA-500 (DMSO d₆) δ 1.366 (t, J=7.0 Hz, 3H), 3.591 (s, 2H), 4.110 (q,J=7.0 Hz, 2H), 4.312 (d, J=5.5 Hz, 2H), 6.985 (d, J=7.5 Hz, 1H),7.048-7.105 (m, 3H), 7.342-7.402 (m, 2H), 7.621 (m, 2H), 7.770 (d, J=7.0Hz, 1H), 7.826 (d, J=8.0 Hz, 1H), 7.942 (d, J=7.5 Hz, 1H), 8.550 (s,1H), 8.701 (s, 1H). MS (m/z) 365.2 (M+H)⁺.

Synthesis of Compound 20,4-{5-[(3-Fluoro-benzylcarbamoyl)-methyl]-pyridin-2-yl}-benzoic Acid,KX1-323

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol. (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis ofN-(3-Fluoro-benzyl)-2-[6-(3-fluoro-phenyl)-pyridin-3-yl]-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.15 gm,0.54 mmole), 4-carboxybenzeneboronic acid (0.096 gm, 0.6 mmole) wasdissolved in DME, Na₂CO₃ (0.11 gm, 1.08 mmole) in 5 ml of water wasadded to the DME solution, the solution was then degassed for 30 min (Arthrough the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.031 gm, 0.027 mmole) was added,degassed for additional 15 min, refluxed for 24 hr. The reaction wasallowed to cool to room temperature, filtered, solid washed with ethylacetate, NaOH 10%, the aqueous layer was washed several times with ethylacetate, neutralized by drop wise addition of HCl 1% having ethylacetate in the medium with shaking after each addition of the HCl, ethylacetate was evaporated and the solid formed was crystallized frommethanol-water to produce a white solid (0.07 gm, 40%). H¹-NMR INOVA-500(DMSO d₆) δ 3.625 (s, 2H), 4.318 (d, J=5.5 Hz, 2H), 7.053-7.111 (m, 3H),7.376 (q, J=7.0 Hz, 1H), 7.8341 (d, J=8.0, 1H), 8.015-8.063 (m, 3H),8.206 (d, J=8.0 Hz, 1H), 8.613 (s, 1H), 8.724 (t, J=5.5, 1H). MS (m/z)365.3 (M+H)⁺.

Synthesis of Compound 21,2-[6-(2-Ethoxy-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide,KX1-324

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol. (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis of2-[6-(2-Ethoxy-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.15 gm,0.54 mmole), 2-ethoxybenzeneboronic acid (0.096 gm, 0.6 mmole) wasdissolved in DME, Na₂CO₃ (0.11 gm, 1.08 mmole) in 5 ml of water wasadded to the DME solution, the solution was then degassed for 30 min (Arthrough the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.031 gm, 0.027 mmole) was added,degassed for additional 15 min, refluxed for 24 hr. The reaction wasallowed to cool to room temperature, filtered, solid washed with ethylacetate; the organic layer was dried, evaporated. The residue waschromatographed using ethyl acetate/hexane 2:1, then it crystallizedfrom methanol-water to produce a white solid (0.075 gm, 40%). H¹-NMRINOVA-500 (DMSO d₆) δ 1.339 (t, J=7.0 Hz, 3H), 3.581 (s, 2H), 4.112 (q,J=7.0 Hz, 2H), 4.322 (d, J=5.5 Hz, 2H), 7.032-7.135 (m, 5H), 7.358-7.387(m, 2H), 7.703 (d, J=7.0, 1H), 7.748 (d, J=7.0 Hz, 1H), 7.871 (d, J=7.0Hz, 1H), 8.548 (s, 1H), 8.725 (s, 1H). MS (m/z) 365.2 (M+H)⁺.

Synthesis of Compound 22,2-[6-(4-Ethoxy-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide,KX1-325

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol. (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis of2-[6-(4-Ethoxy-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.15 gm,0.54 mmole), 4-ethoxybenzeneboronic acid (0.096 gm, 0.6 mmole) wasdissolved in DME, Na₂CO₃ (0.11 gm, 1.08 mmole) in 5 ml of water wasadded to the DME solution, the solution was then degassed for 30 min (Arthrough the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.031 gm, 0.027 mmole) was added,degassed for additional 15 min, refluxed for 24 hr. The reaction wasallowed to cool to room temperature, filtered, solid washed with ethylacetate; the organic layer was dried, evaporated. The residue waschromatographed using ethyl acetate/hexane 2:1, then it crystallizedfrom methanol-water to produce a white solid (0.08 gm, 42%). H¹-NMRINOVA-500 (DMSO d₆) δ 1.357 (t, J=7.0 Hz, 3H), 3.564 (s, 2H), 4.090 (q,J=7.0 Hz, 2H), 4.309 (d, J=6.0 Hz, 2H), 7.012-7.103 (m, 5H), 7.361 (q,J=7.0 Hz, 1H), 7.726 (d, J=8.0 Hz, 1H), 7.842 (d, J=8.0 Hz, 1H), 8.012(d, J=8.5 Hz, 2H), 8.503 (s, 1H), 8.686 (s, 1H). MS (m/z) 365.2 (M+H)⁺.

Scale-Up Synthesis of Compound 22 HCl.2-[6-(4-Ethoxy-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide HCl,KX1-325 HCl

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamideHCl: 2-chloropyridine-5-acetic acid (6.0 gm, 34 mmole),3-fluorobenzylamine (4.5 ml, 34 mmole), PyBOP (18 gm, 36 mmole), andDIEA (12.5 ml, 75 mmole) was dissolved in DMF stirred overnight, thereaction mixture was then poured into water, solid was collected byfiltration, re-crystallized using water-methanol (6.3 gm, 70%); H¹-NMRINOVA-500 (CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s,1H), 6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz,1H), 8.280 (d, J=2.5 Hz, 1H).

Synthesis of2-[6-(4-Ethoxy-phenyl)-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (4.8 gm,17.2 mmole), 4-ethoxybenzeneboronic acid (3.14 gm, 18.9 mmole) wassuspended in DME (100 ml), Na₂CO₃ (3.6 gm, 34.4 mmole) in 15 ml of waterwas added to the DME solution, the solution was then degassed for 30 min(Ar through the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.99 gm, 0.86 mmole) was added,degassed for additional 15 min, refluxed overnight. The reaction wasallowed to cool to room temperature, filtered, the solid washed withcold ethyl acetate and saturated NaHCO₃ solution, the solid was thenrecrystallized from methanol to produce white solid (4.8 gm).

4.6 gm of the free amine was dissolved in 50 ml ethanol with gentleheating, then 25 ml of 4 N HCl in ethyl acetate was added, the solutionwas concentrated to 20 ml, then diluted with 100 ml of cold ethylacetate, the solid formed was filtered washed with more ethyl acetate(50×2) and dried (4.3 gm, 65%); H¹-NMR INOVA-500 (DMSO d₆) δ 1.386 (t,J=7.0 Hz, 3H), 3.822 (s, 2H), 4.179 (q, J=7.0 Hz, 2H), 4.339 (d, J=6.0Hz, 2H), 7.074-7.182 (m, 5H), 7.374 (m, 1H), 8.106 (d, J=8.0 Hz, 1H),8.263 (d, J=8.0 Hz, 1H), 8.312 (s, 2H), 8.718 (s, 1H), 8.981 (s, 1H). MS(m/z) 365.2 (M+H)⁺.

Melting Point of the free base: 0.1 gm of the HCl salt was stirred in 10ml of 20% NaOH for 10 min, filtered; the solid was crystallized fromethanol water, dried in the oven at 100° C. for 2 hrs. Melting point wasfound to be 173-176° C.

Synthesis of Compound 23,N-(3-Fluoro-benzyl)-2-[6-(4-methanesulfonyl-phenyl)-pyridin-3-yl]-acetamide,KX1-326

Synthesis of 2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide:2-chloropyridine-5-acetic acid (0.2 gm, 1.21 mmole), 3-fluorobenzylamine(0.15 ml, 1.2 mmole), PyBOP (0.67 gm, 1.3 mmole), and DIEA (0.43 ml, 2.6mmole) was dissolved in DMF stirred overnight, the reaction mixture wasthen poured into water, solid was collected by filtration,re-crystallized using water-methanol (0.3 gm, 85%); H¹-NMR INOVA-500(CDCl₃) δ 3.562 (s, 2H), 4.429 (d, J=6.5 Hz, 2H), 5.868 (s, 1H),6.929-7.015 (m, 3H), 7.300-7.333 (m, 2H), 7.668 (dd, J=8, 2.5 Hz, 1H),8.280 (d, J=2.5 Hz, 1H).

Synthesis ofN-(3-Fluoro-benzyl)-2-[6-(4-methanesulfonyl-phenyl)-pyridin-3-yl]-acetamide:2-(6-Chloro-pyridin-3-yl)-N-(3-fluoro-benzyl)-acetamide and (0.15 gm,0.54 mmole), 4-methanesulfonyl benzeneboronic acid (0.12 gm, 0.6 mmole)was dissolved in DME, Na₂CO₃ (0.11 gm, 1.08 mmole) in 5 ml of water wasadded to the DME solution, the solution was then degassed for 30 min (Arthrough the solution and vacuum applied for the first 5 min),Palladiumtetrkestriphenylphosphine (0.031 gm, 0.027 mmole) was added,degassed for additional 15 min, refluxed for 24 hr. The reaction wasallowed to cool to room temperature, filtered, solid washed with ethylacetate; the organic layer was dried, evaporated. The residue waschromatographed using ethyl acetate/hexane 2:1, then it crystallizedfrom methanol-water to produce a white solid (0.02 gm, 10%); H¹-NMRINOVA-500 (DMSO d₆) δ 3.341 (s, 3H), 3.635 (s, 2H), 4.315 (d, J=7.0 Hz,2H), 7.047-7.110 (m, 3H), 7.366 (q, J=9.0 Hz, 1H), 7.857 (d, J=8.5 Hz,1H), 8.027-8.081 (m, 3H), 8.343 (d, J=10.5 Hz, 2H), 8.631 (s, 1H), 8.731(s, 1H). MS (m/z) 399.2 (M+H)⁺.

Synthesis of Compound 24, KX1-327, and Compound 26, KX1-357

The syntheses are shown in Scheme 5.

Compound 24, KX1-327 HCl

A solution of 75 mL 1,2-Dimethoxyethane and 16 mL 2 M sodium carbonatewas thoroughly degassed by heating at 50° C. with an argon streamthrough the solvent. 5.00 g of the 4-bromophenyl acetamide (5, 15.6mmol) and 1.95 grams of 4-pyridylboronic acid (1.00 eq) were added toand degassing continued for 1 hour. Tetrakis(triphenylphosphine)palladium (5 mol %) was added neat and the reaction was refluxed for 24hours. The reaction was cooled and poured into 300 mL distilled waterand filtered to give 5.014 g crude product. This crude product was takenup in 1 L of a 1 to 1 mix of 1 N HCl and ethyl acetate. The organiclayer was discarded and the aqueous layer was washed two more times withEtOAc. The aqueous layer was the basified with solid sodium bicarbonateto pH 7.5. This was then extracted 3×300 mL EtOAc to give about 3.25 gof semi-pure product. Pure crystals of the free base were made bydissolving 200 mg in a minimum amount of ethyl acetate with gentleheating and sonication. Hexanes was added to this solution until itbecame cloudy. This was heated until clear. Addition of more hexanesfollowed by heating was repeated two more times. This clear solution wasallowed to stand overnight in a sealed vessel. White crystals formedwhich were washed with hexanes and dried to give about 50 mg (mp145-146° C.). The rest of the product was dissolved in ethanol and twoequivalents of hydrochloric acid (1.1 M in EtOAc) were added. After 1hour the ethanol was removed and redissolved in the least amount ofethanol at 40° C. EtOAc was added until the solution became cloudy. Thesolution was allowed to stand and the desired product crystallized aspure white crystals. The crystals were filtered off, washed with EtOAcand dried to give 2.4 grams (48% overall yield); LCMS 321.3 (m+H). ¹HNMR (500 MHz, DMSO) 3.61 (s, 2H) 4.29 (d, 7.5 Hz, 2H) 7.04 (m, 3H) 7.34(q, 9.5 Hz, 1H) 7.50 (d, 10.5 Hz, 2H) 7.95 (d, 10.5 Hz, 2H) 8.24 (d, 8.0Hz, 2H) 8.70 (s. 1H) 8.87 (d, 8.0 Hz, 2H).

Compound 26, KX1-357

47.0 mg of KX1-327 were dissolved in 5 mL DCM. Meta-chloroperoxybenzoicacid (35.0 mg, 1.4 eq) were added and the reaction was allowed to stirfor 13 hours. The reaction was washed 3×5 mL saturated sodiumbicarbonate, dried with sodium sulfate and concentrated to give 45 mg ofa yellow solid. NMR revealed the product contained about 15% impurity,which may have been m-chlorobenzoic acid (or the peroxide). The solidwas redissolved in 5 mL DCM and washed 3×5 mL saturated sodiumbicarbonate, dried with sodium sulfate and concentrated to give 26 mg ofthe desired product as a yellow solid; LCMS 337.2 (M+H), 672.9 (2M+H),694.8 (2M+Na). ¹H NMR (400 MHz, DMSO) 3.54 (s, 2H), 4.28 (d, 6.0 Hz,2H), 7.00-7.08 (m, 3H), 7.34 (q, 8.0 Hz, 1H), 7.40 (d, 8.4 Hz, 2H), 7.72(d, 8.4 Hz, 2H), 7.75 (d, 7.2 Hz, 2H), 8.24 (d, 8.4 Hz, 2H), 8.63 (t,5.6 Hz, 1H).

4-Bromophenylacetic acid (6.00 g, 47.9 mmol) was dissolved in 40 mL ofanhydrous dichloromethane under an argon atmosphere and cooled in an icebath. 3-Fluorobenzylamine (1.00 eq) was added and unintendedprecipitation of the acetic acid/benzylamine salt occurred. Moredichloromethane (20 mL) was added followed by DIEA (2.2 eq), HOBT (1.0eq), and EDCI (1.1 eq). After about 2 hours the solid broke up, 4 hoursafter that the reaction was finished by TLC. The reaction was dilutedwith 200 mL of dichloromethane and 200 mL of 1 N hydrochloric acid. Uponshaking in a separatory funnel an emulsion formed. This emulsion wasdivided in half and dichloromethane was removed. 500 mL ethyl acetateand another 300 mL 1 N HCl was added to each half. The organic layer waswashed 2 more times with 1 N HCl, 3×300 mL saturated sodium bicarbonate,and 3×200 mL with saturated sodium chloride. Organic layers from eachextraction were combined and dried with sodium sulfate, and solvent wasremoved to give 13.12 g (85% yield) desired product; ¹H NMR (500 MHz,CDCl₃) δ (ppm) 3.58 (s, 2H), 4.45 (d, 6.0 Hz, 2H), 5.70 (bs, 1H) 6.93(m, 3H), 7.16 (d, 8.1 Hz, 2H), 7.26 (m, 1H) 7.48 (d, 8.1 Hz, 2H).

Synthesis of Compound 25, KX1-329

As shown in Scheme 6, 5-Hydroxy-2-methylpyridine was converted to thetriflate, 6, followed by Suzuki reaction to give the5-phenyl-2-methylpyridine. The methyl pyridine, 7, was deprotonated withn-butyllithum and added to a solution of ethyl carbonate. Saponificationfollowed by amide coupling with PyBOP gave the desired product.

5-Hydroxy-2-methylpyridine (3.00 g, 27.5 mmol) was dissolved in 15 mLanhydrous pyridine and cooled to 0° C. Triflic anhydride (7.76 g, 1.1eq) was added drop wise over 3 minutes. Following the addition thereaction was removed from the ice bath and allowed to stir for 6 hr. Thevolume was then reduced to 8 mL in vacuo, diluted with 50 mL distilledwater, and then extracted with 75 mL EtOAc. The organic layer was thenwashed with 1 N HCl (3×50 mL), dried with sodium sulfate, and removed invacuo to give 2.78 g (42%) of an amber oil (6); LCMS 242.1 (m+H). ¹H NMR(400 MHz, CDCl₃) 2.58 (s, 3H) 7.26 (d, 8.4 Hz, 1H) 7.52 (dd, 2.8 Hz, 8.4Hz, 1H) 8.47 (d, 2.8 Hz, 1H).

A flame dried 50 mL round bottom flask with two condensers was chargedwith argon. Dimethoxyethane, 25 mL and 6 mL 2 M sodium carbonate washeated to 45° C. while argon was bubbled through the solution. After 1hour, the pyridyl triflate (6) (1.538 g, 6.382 mmol) and phenylboronicacid (856 mg, 1.1 eq) were added. After one hour Pd(PPh₃)₄ (370 mg, 5mol %) was added, the reaction was heated at 65-75° C. for 48 hours. Thesolvent was poured into a round bottom flask, the remaining residue waswashed with ethyl acetate. Solvents were combined and removed in vacuo.The residue was purified by silica gel chromatography (hexanes:EtOAc) togive 702 mg of the desired product 7 (65% yield); LCMS 170.2 (m+H). ¹HNMR (400 MHz, CDCl₃) 3.60 (s, 3H) 7.22 (d, 8.0 Hz, 1H) 7.38 (t, 7.2 Hz,1H) 7.46 (t, 7.2 Hz, 2H) 7.56 (d, 8.0 Hz, 2H) 7.77 (dd, 2.4 Hz, 8.0 Hz,1H) 8.73 (d, 2.4 Hz, 1H).

5-Phenyl-2-methylpyridine (7, 205 mg, 1.223 mmol) was dissolved infreshly distilled THF in flame dried glassware under argon. Cooled to−78° C. in a dry ice/acetone bath for 20 minutes. N-Butyllithium (0.485mL, 1.0 eq) was added drop wise over 5 minutes. This solution was addedto a THF solution of ethyl carbonate (1.5 eq) via a cannula. Thesolution was stirred for 2 hours before being quenched with methanoladded drop wise. 1 N sodium hydroxide (1 mL) was added before removingthe organic solvents in vacuo. The remaining aqueous solution wasextracted with ether (3×15 mL). Organic layers were combined and driedwith sodium sulfate and removed in vacuo to give 208 mg 8 (71% yield) ¹HNMR (500 MHz, CDCl₃) 1.30 (m, 3H) 2.61 (s, 2H) 4.20 (m, 3H) 7.22 (d, 8.0Hz, 1H) 7.38 (t, 7.5 Hz, 1H) 7.48 (t, 7.5 Hz, 2H) 7.58 (m, 2H) 7.78 (dd,2.5 Hz, 8.0 Hz, 1H) 8.73 (d, 2.5 Hz, 1H).

Ethyl ester 8 (208 mg, 0.86 mmol) was dissolved in 5 mL THF. 1 N NaOH(about 1 mL) was added and the reaction was put in a 35° C. water bathovernight. The volume of the reaction was reduced to about 1 mL and thenacidified with 1 N HCl to precipitate the desired product. Theprecipitate was isolated by decanting and drying in vacuo to give 54 mg(30% yield) of 9; LCMS 214.1 (m+H) 236.0 (m+Na). ¹H NMR (400 MHz, CD₃OD)3.64 (s, 2H) 7.24-7.28 (m, 4H) 7.25 (t, 8.4 Hz, 2H) 7.52 (d, 8.4 Hz, 2H)7.87 (dd, 2.0 Hz, 8.0 Hz, 1H) 8.53 (d, 2.0 Hz, 1H).

Carboxylic acid 9 (54 mg, 0.232 mmol), 3-Fluorobenzylamine (1.1 eq), andPyBOP (1.1 eq) were dissolved in 3 ml anhydrous DMF. After 10 minutesDIEA (1.1 eq) was added and the reaction was allowed to stir overnight.The DMF was removed in vacuo and the residue was taken up with methanoland crystallized from methanol/water to give 44 mg Compound 25, KX1-329(55%) as clear, needle crystals; TLC, Rf 0.2 (1:1DCM:EtOAc). LCMS 321.2(m+H), 343.1 (m+Na), 662.9 (2m+Na). ¹H NMR (400 MHz, CDCl₃) 3.82 (s,2H), 4.46 (d, 8.8 Hz, 2H), 6.91 (t, 9.2 Hz, 2H) 6.99 (d, 7.6 Hz, 1H),7.25 (t, 8.4 Hz, 2H), 7.34 (d, 8.0 Hz, 2H) 7.40 (tt, 1.2 Hz, 7.2 Hz, 2H)7.55 (d, 7.6 Hz, 2H) 7.80 (b, 1H) 7.86 (dd, 2.0 Hz, 7.6 Hz, 1H) 8.73 (d,2.0 Hz, 1H).

Synthesis of Compound 27,2-[6-(4-Ethoxy-phenyl)-1-oxo-pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide,KX1-358

To an ice cooled solution of 0.2 gm of 2-[6-(4-Ethoxy-phenyl)pyridin-3-yl]-N-(3-fluoro-benzyl)-acetamide in 80 ml DCM, 0.13 gm ofm-chloroperbenzoic acid was added as solid. After stirring overnight,the reaction was washed with saturated sodium bicarbonate solution,dried with sodium sulfate, evaporated to dryness under vacuum, thenchromatographed (silica gel) using ethyl acetate followed by 10%methanol in ethyl acetate to produce 0.16 gm (78%); H¹-NMR INOVA-400(DMSO d₆) δ 1.357 (t, J=7.0 Hz, 3H), 3.564 (s, 2H), 4.090 (q, J=6.8 Hz,2H), 4.309 (d, J=5.60 Hz, 2H), 7.012-7.103 (m, 5H), 7.245 (d, J=8.0 Hz,1H), 7.729 (m, 1H), 7.529 (d, J=8.0 Hz, 1H), 7.800 (d, J=8.5 Hz, 2H),8.225 (s, 1H), 8.663 (t, J=5.6 Hz, 1H). MS (m/z) 380 (M+H)⁺.

For the following syntheses, unless otherwise noted, reagents andsolvents were used as received from commercial suppliers. Proton andcarbon nuclear magnetic resonance spectra were obtained on a Bruker AC300 or a Bruker AV 300 spectrometer at 300 MHz for proton and 75 MHz forcarbon. Spectra are given in ppm (S) and coupling constants, J, arereported in Hertz. Tetramethylsilane was used as an internal standardfor proton spectra and the solvent peak was used as the reference peakfor carbon spectra. Mass spectra and LC-MS mass data were obtained on aPerkin Elmer Sciex 100 atmospheric pressure ionization (APCI) massspectrometer. LC-MS analyses were obtained using a Luna C₈ (2) Column(100×4.6 mm, Phenomenex) with UV detection at 254 nm using a standardsolvent gradient program (Method B). Thin-layer chromatography (TLC) wasperformed using Analtech silica gel plates and visualized by ultraviolet(UV) light, iodine, or 20 wt % phosphomolybdic acid in ethanol. HPLCanalyses were obtained using a Prevail C18 column (53×7 mm, Alltech)with UV detection at 254 nm using a standard solvent gradient program(Method A).

Time Flow (min) (mL/min) % A % B  0.0 3.0 95.0  5.0 10.0 3.0  0.0 100.011.0 3.0  0.0 100.0 A = Water with 0.1 v/v Trifluoroacetic Acid B =Acetonitrile with 0.1 v/v Trifluoroacetic Acid

Time Flow (min) (mL/min) % A % B 0.0 2.0 95.0  5.0 4.0 2.0  5.0 95.0 A =Water with 0.02 v/v Trifluoroacetic Acid B = Acetonitrile with 0.02 v/vTrifluoroacetic Acid

Synthesis of N-benzyl-2-(5-bromopyridin-2-yl)acetamide

A flask was charged with 5-(5-bromopyridin-2(1H)-ylidene)-2,2-dimethyl-1,3-dioxane-4,6-dione (1.039 g, 3.46 mmol),benzylamine (0.50 mL, 4.58 mmol), and toluene (20 mL). The reaction wasbrought to reflux under nitrogen for 18 hours, then cooled and placed ina freezer until cold. The product was collected by filtration and washedwith hexanes to yield a mass of bright white crystals (1.018 g, 96%).

Synthesis of 4-(2-(4-(4,4,5,5-tetramethyl[,3,2]dioxaborolan-2-yl)-phenoxy)ethyl)morpholine

To a stirring solution of4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-phenol (2.55 g, 11.58mmol), 2-morpholin-4-ylethanol (1.60 mL, 1.73 g, 13.2 mmol) andtriphenyl phosphine (3.64 g, 13.9 mmol) in methylene chloride (60 mL) at0° C. was added dropwise DIAD (2.82 g, 13.9 mmol). The reaction wasallowed to warm to room temperature and stir overnight. After 18 hours,additional portions of triphenyl phosphine (1.51 g, 5.8 mmol),2-morpholin-4-ylethanol (0.70 mL, 5.8 mmol), and DIAD (1.17 g, 5.8 mmol)were added. After stirring an additional 2 hours at room temperature thereaction was concentrated and the residue purified by flashchromatography (5% to 25% EtOAc in CHCl₃) to provide the product as awhite solid (2.855 g, 74%).

Synthesis of Compound 134, KX2-391 or KXO1

A 10 mL reaction tube with a septum closure and stir bar was chargedwith N-benzyl-2-(5-bromopyridin-2-yl)acetamide (123 mg, 0.403 mmol),4-(2-(4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)-phenoxy)ethyl)morpholine(171 mg, 0.513 mmol), and FibreCat 1007¹ (30 mg, 0.015 mmol). Ethanol (3mL) was added, followed by aqueous potassium carbonate solution (0.60mL, 1.0 M, 0.60 mmol). The tube was sealed and heated under microwaveconditions at 150° C. for 10 minutes. The reaction was cooled andconcentrated to remove the majority of the ethanol, and then taken up in10 mL of ethyl acetate and washed successively with water and saturatedsodium chloride solution. The organic layer was dried with MgSO₄,filtered and concentrated to a white solid. This white solid wastriturated with ethyl ether to give ALB 30349 as a white solid (137 mg,79%): mp 135-137° C.; ¹H NMR (300 MHz, CDCl₃) δ 8.70 (d, 1H, J=2.0 Hz),7.81 (dd, 1H, J=2.4 Hz, J=8.0 Hz), 7.65 (br s, 1H), 7.49 (d, 2H, J=8.8Hz), 7.37-7.20 (m, 6H), 7.01 (d, 2H, J=8.8 Hz), 4.49 (d, 2H, J=5.8 Hz),4.16 (t, 2H, J=5.7 Hz, 3.82 (s, 2H), 3.78-3.72 (m, 4H), 2.84 (t, 2H,J=5.7 Hz), 2.62-2.58 (m, 4H); HPLC (Method B) 98.0% (AUC), t_(R)=1.834min.; APCI MS m/z 432 [M+H]⁺. ¹Polymer bounddi(acetato)dicyclohexylphenylphosphinepalladium(II), manufactured byJohnson Matthey, Inc. and available from Aldrich (catalog #590231).

(4-bromo-3-fluorophenyl)(morpholino)methanone

A 500 mL flask was charged with 4-bromo-3-fluorobenzoic acid (5.00 g,22.83 mmol), 100 mL DMF, morpholine (2.4 ml, 27.5 mmol), and4-Ethylmorpholine (8.6 ml, 67.9 mmol). HOBt (4.32 g, 32.0 mmol) wasadded followed by EDC (5.25 g, 27.4 mmol) and the reaction allowed tostir at room temperature for 18 hours. The reaction was concentrated andthe resulting orange syrup taken up in 100 mL EtOAc and 100 mL water.The organic layer was washed with 100 mL 2N HCl, 100 mL saturated sodiumbicarbonate, and 100 mL saturated sodium chloride. The organic was thendried with MgSO₄, filtered, and concentrated to give 6.476 g (98%) of aviscous yellow oil. This material was used without further purification.

4-(4-bromo-3-fluorobenzyl)morpholine

A 250 ml flask was charged with(4-bromo-3-fluorophenyl)(morpholino)methanone (4.569 g, 15.86 mmol) anddissolved in 16 mL of THF. Diphenylsilane (6.2 ml, 33.4 mmol) was addedfollowed by carbonyltris(triphenylphosphine)rhodium(I)hydride (100 mg,0.109 mmol) and the reaction stirred at room temperature for 20 hours.

The reaction was diluted with 200 mL of ether and extracted with 1N HCl(2×150 mL). This resulted in the formation of a white precipitate in theseparatory funnel. The acid layer and the resulting white precipitatewere washed with ether (2×100 mL), and then basified with solid NaOHpellets (23 g). The aqueous layer was then extracted with ether (3×125mL), dried over MgSO₄, filtered, and concentrated to give 1.35 g (31%)of a colorless oil. This material was used without further purification.

4-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)morpholine

A 10 mL microwave reaction tube with septum closure was charged with4-(4-bromo-3-fluorobenzyl)morpholine (405 mg, 1.48 mmol),Bis(pinacolato)diboron (516 mg, 2.03 mmol), Pd(dppf)Cl₂.CH₂Cl₂ (62 mg,0.076 mmol), potassium acetate (659 mg, 6.72 mmol), and DMF (3.6 mL).The vial was placed under nitrogen by evacuation/backfilling (5 cycles)and stirred at 80° C. for 8 hours. The reaction was cooled, diluted withethyl acetate (25 mL) and filtered. The organics were washed with water(25 mL) and saturated sodium chloride (25 mL). The organic layer wasthen dried over MgSO₄ and concentrated to a dark oil. The product waspurified by silica gel chromatography eluting with 2% MeOH in CHCl₃ togive 310 mg (65%) of an off-white solid.

Synthesis of Compound 136, KX2-393

A 10 mL microwave reaction tube with septum closure was charged with4-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)morpholine(307 mg, 0.96 mmol), 2-(5-bromopyridin-2-yl)-N-(3-fluorobenzyl)acetamide(247 mg, 0.77 mmol), and FibreCat 1007 (60 mg, 0.03 mmol). Ethanol (3mL) was added followed by aqueous potassium carbonate solution (1.2 mL,1.0 M, 1.2 mmol). The tube was sealed and heated under microwaveconditions at 150° C. for 10 minutes. The reaction was cooled andconcentrated to remove the majority of the ethanol, and then taken up in10 mL of ethyl acetate and washed successively with water and saturatedsodium chloride solution. The organic layer was dried with MgSO₄,filtered, and concentrated. The material was purified by columnchromatography (silica gel, 100:0 CHCl₃/MeOH to 95:5 CHCl₃/MeOH) toprovide ALB 30351 as a white solid (240 mg, 74%): mp 91-92° C.; ¹H NMR(300 MHz, CDCl₃) δ 8.71 (br s, 1H), 7.86-7.84 (m, 1H), 7.78 (br s, 1H),7.37 (t, 2H, J=7.5 Hz), 7.28-7.21 (m, 3H), 7.02 (dd, 1H, J=0.6 Hz, J=7.7Hz), 6.98-6.90 (m, 2H), 4.49 (d, 2H, J=5.9 Hz), 3.84 (s, 2H), 372-3.75(m, 4H), 3.52 (s, 2H), 2.47-2.50 (m, 4H); HPLC (Method A) 98.7% (AUC),t_(R)=3.866 min.; APCI MS m/z 438 [M+H]⁺.

4-(2-(4-bromo-3-fluorophenoxy)ethyl)morpholine

A flask was charged with 4-bromo-3-fluorophenol (4.999 g, 26.2 mmol) andtriphenylphosphine (10.298 g, 39.3 mmol). Methylene chloride (120 mL)was added followed by 2-morpholinoethanol (4 mL, 33.0 mmol) and thesolution was stirred on an ice water bath to cool. After 5 minutes,diisopropyl azodicarboxylate (7.6 ml, 39.1 mmol) was added over 6 to 8minutes. The reaction was left stirring on the cold bath to slowly warmto room temperature overnight. The reaction was concentrated and theresidue purified by flash chromatography (25% to 100% EtOAc in hexanes)to provide the product as a colorless oil (2.621 g, 33%).

4-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)morpholine

A 40 mL microwave reaction tube with a septum closure and stir bar wascharged with 4-(2-(4-bromo-3-fluorophenoxy)ethyl)morpholine (307 mg, 1.0mmol), Bis(pinacolato)diboron (318 mg, 1.25 mmol), Pd(dppf)Cl₂—CH₂Cl₂(68 mg, 83 μmol), and Potassium acetate (316 mg, 3.22 mmol). DME (20 ml)was added and the tube sealed. The tube was evacuated/backfilled w. N2(5 cycles) and microwaved at 125° C. for 30 minutes. The reaction wascooled to room temperature, concentrated and the residue purified bycolumn chromatography (silica gel, 2% MeOH in CHCl₃) to provide theproduct as a colorless oil (356 mg, >99%). The ¹H NMR spectrum shows theproduct to contain a small amount of pinacol-like impurity. The materialwas used as-is.

Synthesis of Compound 133, KX2-392

A 10 mL microwave reaction tube with septum closure was charged with4-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)morpholine(175 mg, 0.50 mmol), 2-(5-bromopyridin-2-yl)-N-(3-fluorobenzyl)acetamide(121 mg, 0.37 mmol), and FibreCat 1007 (30 mg, 0.03 mmol). Ethanol (3mL) was added followed by aqueous potassium carbonate solution (0.600mL, 1.0 M, 0.60 mmol). The tube was sealed and heated under microwaveconditions at 150° C. for 10 minutes. The reaction was cooled, filtered,and concentrated to remove the majority of the ethanol. The residue wasthen taken up in 10 mL of ethyl acetate and washed successively withwater and saturated sodium chloride solution. The organic layer wasdried with MgSO₄, filtered, and concentrated. The material was purifiedby column chromatography (silica gel, 100:0 CHCl₃/MeOH to 95:5CHCl₃/MeOH) to provide ALB 30350 as a white solid (70 mg, 40%): mp126-127° C.; ¹H NMR (500 MHz, CDCl₃) δ 8.67 (br s, 1H), 7.77-7.85 (m,2H), 7.21-7.37 (m, 3H), 7.02 (d, 1H, J=7.7 Hz), 6.90-6.97 (m, 2H), 6.82(dd, 1H, J=2.5 Hz, J=8.6 Hz), 6.76 (dd, 1H, J=2.4 Hz, J=12.4 Hz), 4.49(d, 2H, J=5.9 Hz), 4.15 (t, 2H, J=5.7 Hz), 3.83 (s, 2H), 3.71-3.78 (m,4H), 2.83 (t, 2H, J=5.7 Hz), 2.56-2.63 (m, 4H); HPLC (Method A)>99%(AUC), t_(R)=4.026 min.; APCI MS m/z 468 [M+H]⁺.

1-(2-(4-bromo-3-fluorophenoxy)ethyl)-4-methylpiperazine

A flask was charged with 4-bromo-3-fluorophenol (5.00 g, 26 mmol) andtriphenylphosphine (10.30 g, 39 mmol). Methylene chloride (120 mL) wasadded followed by 2-(4-methylpiperazin-1-yl)ethanol (4.61 g, 32 mmol)and the solution was stirred on an ice water bath to cool. After 5minutes, diisopropyl azodicarboxylate (7.6 ml, 39.1 mmol) was added over6 to 8 minutes. The reaction was left stirring on the cold bath toslowly warm to room temperature overnight. The reaction was concentratedand the residue purified by flash chromatography (25% to 100% EtOAc inhexanes) to provide the product as a colorless oil (2.62 g, 33%).

1-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)-4-methylpiperazine

A 40 mL microwave reaction tube with a septum closure and stir bar wascharged with 1-(2-(4-bromo-3-fluorophenoxy)ethyl)-4-methylpiperazine(428 mg, 1.35 mmol), Bis(pinacolato)diboron (375 mg, 1.48 mmol),Pd(dppf)Cl₂—CH₂Cl₂ (63 mg, 77 μmol), and Potassium acetate (410 mg, 4.18mmol). DME (10 ml) was added and the tube sealed. The tube wasevacuated/backfilled w. N₂ (5 cycles) and microwaved at 100° C. for 30minutes. Additional Pd(dppf)Cl₂—CH₂Cl₂ (63 mg, 77 μmol) was added andthe reaction microwaved at 100° C. for 60 minutes. The reaction wascooled to room temperature, concentrated and the residue purified bycolumn chromatography (silica gel, 1% to 2% MeOH in CHCl₃) to providethe product as a dark oil (354 mg, 72%).

Synthesis of Compound 137, KX2-394

A 10 mL microwave reaction tube with septum closure was charged with1-(2-(3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)-4-methylpiperazine(340 mg, 0.93 mmol), 2-(5-bromopyridin-2-yl)-N-(3-fluorobenzyl)acetamide(201 mg, 0.62 mmol), and FibreCat 1007 (125 mg, 0.06 mmol). Ethanol (3mL) was added followed by aqueous potassium carbonate solution (1.00 mL,1.0 M, 1.00 mmol). The tube was sealed and heated under microwaveconditions at 150° C. for 10 minutes. The reaction was cooled, filtered,and concentrated to remove the majority of the ethanol. The residue wasthen taken up in 10 mL of ethyl acetate and washed successively withwater and saturated sodium chloride solution. The organic layer wasdried with MgSO₄, filtered, and concentrated. The material was purifiedby column chromatography (silica gel, 98:2 CHCl₃/MeOH to 90:10CHCl₃/MeOH) to provide ALB 30352-2 as a tan gum (28 mg, 9%): ¹H NMR (300MHz, CDCl₃) δ 8.66 (br s, 1H), 7.78-7.94 (m, 2H), 7.20-7.40 (m, 3H),6.88-7.06 (m, 3H), 6.70-6.85 (m, 2H), 4.47 (d, 2H, J=5.9 Hz), 4.14 (t,2H, J=5.7 Hz), 3.83 (s, 2H), 2.85 (t, 2H, J=5.7 Hz), 2.41-2.77 (m, 8H),2.34 (s, 3H); HPLC (Method A)>99% (AUC), t_(R)=3.778 min.; APCI MS m/z481 [M+H]⁺.

Synthesis of Compound 139 (KX2-402) di-HCl Salt

Step 1:

Into a 500 ml 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placed a solution of3-fluorobenzonitrile (25 g, 206.61 mmol, 1.00 equiv) in THF (300 ml). Tothe mixture was added LiAlH₄ (7.7 g, 202.63 mmol, 0.98 equiv), whilecooling to a temperature of 0° C. The resulting solution was allowed toreact, with stirring, for 3 hours while the temperature was maintainedat 0° C. The reaction progress was monitored by TLC (EtOAc/PE=1:3). Thereaction mixture was then quenched by the adding 7.7 ml of H₂O, 7.7 mlof 2M NaOH/H₂O and 21 ml of H₂O. A filtration was performed. Thefiltrate was concentrated by evaporation under vacuum using a rotaryevaporator. This resulted in 12 g (42%) of (3-fluorophenyl)methanamineas a yellow oil.

LC-MS—PH—NC—KX-2-402-20: (ES, m z): N/A

H-NMR—PH—NC—KX-2-402-20: (CDCl₃, ppm): N/A

Step 2:

Into a 500 ml 3-necked round-bottom flask, was placed a solution of2-(4-bromophenyl)acetic acid (25 g, 116.28 mmol, 1.00 equiv) in THF (300ml). To this was added CDI (20.3 g, 125.31 mmol, 1.08 equiv). Themixture was stirred for 30 minutes at room temperature. To the mixturewas added (3-fluorophenyl)methanamine (17.2 g, 137.53 mmol, 1.18 equiv).The resulting solution was allowed to react, with stirring, for 1 hourwhile the temperature was maintained at 15° C. The reaction mixture wasthen quenched by adding 500 ml of H₂O. The resulting solution wasextracted three times with 200 ml of EtOAc and the organic layerscombined and dried over Na₂SO₄ and concentrated by evaporation undervacuum using a rotary evaporator. This resulted in 12 g (29%) ofN-(3-fluorobenzyl)-2-(4-bromophenyl)acetamide as a yellow solid.

LC-MS: (ES, m z): N/A

H-NMR: (400 MHz, d₆-DMSO) δ 3.468 (1H, s), 4.26-4.27 (2H, s), 6.99-7.06(3H, m), 7.22-7.24 (2H, m), 7.31-7.36 (1H, m), 7.48-7.50 (2H, m), 8.60(1H, s).

Step 3:

Into a 250 ml 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placedN-(3-fluorobenzyl)-2-(4-bromophenyl)acetamide (10 g, 31.04 mmol, 1.00equiv). To this was added4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane(9 g, 35.44 mmol, 1.14 equiv) followed by addition of KOAc (9 g, 91.71mmol, 2.95 equiv) and Pd(dppf)Cl₂ (1 g). To the mixture was added DMF(100 ml). The resulting solution was allowed to react, with stirring,for 24 hours while the temperature was maintained at 85° C. The reactionprogress was monitored by TLC (eluted with EtOAc). The reaction mixturewas cooled to 15° C. in a bath of H₂O. The reaction mixture was thenquenched by adding 500 ml of H₂O. The resulting solution was extractedthree times with 100 ml of EtOAc and the organic layers combined anddried over Na₂SO₄. A filtration was performed and the filtrate wasconcentrated by evaporation under vacuum using a rotary evaporator. Theresidue was purified by eluting through a silica gel column with a 1:30EtOAc/PE solvent system. This resulted in 8 g (70%) ofN-(3-fluorobenzyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamideas a white solid.

LC-MS: [M+H]⁺ calcd for C₂₁H₂₆BFNO₃: 370, found: 370.

H-NMR: (400 MHz, d₆-DMSO) δ 1.29 (12H, s), 3.52 (2H, s), 4.27-4.29 (2H,m), 7.00-7.07 (3H, m), 7.29-7.37 (3H, m), 7.61-7.62 (2H, m), 8.61 (1H,s).

Step 4:

Into a 1000 ml 3-necked round-bottom flask, was placed a solution of4-bromo-3-fluorophenol (40 g, 210.53 mmol, 1.00 equiv) in DMF (400 ml).To this was added 2-bromo-1,1-diethoxyethane (45 g, 228.43 mmol, 1.06equiv), followed by K2CO₃ (35 g, 253.26 mmol, 1.20 equiv). The resultingsolution was allowed to react, with stirring, for 16 hours while thetemperature was maintained at 80° C. The reaction progress was monitoredby TLC (EtOAc/PE=1:5). The reaction mixture was then quenched by adding1500 ml of H₂O. The resulting solution was extracted three times with500 ml of EtOAc and the organic layers combined. The resulting mixturewas washed once with 500 ml of brine. The mixture was dried over Na₂SO₄and concentrated by evaporation under vacuum using a rotary evaporator.This resulted in 50 g (70%) of1-bromo-4-(2,2-diethoxyethoxy)-2-fluorobenzene as a yellow oil.

LC-MS: (ES, m z): N/A

H-NMR: (CDCl₃, ppm): N/A

Step 5:

Into a 500 ml 3-necked round-bottom flask, was placed a solution of1-bromo-4-(2,2-diethoxyethoxy)-2-fluorobenzene (50 g, 162.87 mmol, 1.00equiv) in THF (300 ml)/HCl (30 ml, 65%). The resulting solution wasallowed to react, with stirring, for 2 hours while the temperature wasmaintained at 20° C. The reaction progress was monitored by TLC(EtOAc/PE=1:3). The reaction mixture was then quenched by adding 500 mlof H₂O. Adjustment of the pH to 7-8 was accomplished by the addition ofNa₂CO₃ saturated aqueous solution. The resulting solution was extractedthree times with 300 ml of EtOAc and the organic layers combined. Theresulting mixture was washed once with 300 ml of brine. The mixture wasdried over Na₂SO₄. A filtration was performed. The filtrate wasconcentrated by evaporation under vacuum using a rotary evaporator. Theresidue was purified by eluting through a column with a 1:100 to 1:30EtOAc/PE solvent system. This resulted in 20 g (52%) of2-(4-bromo-3-fluorophenoxy)acetaldehyde as a yellow oil.

GC-MS: [M+H]⁺ calcd for C₈H₇BrFO₂: 233, found: 233.

H-NMR: (400 MHz, CDCl₃) δ 4.57 (2H, s), 6.60-6.62 (1H, m), 6.70-6.72(1H, m), 7.43-7.47 (1H, m), 9.83 (1H, s).

Step 6:

Into a 500 ml 3-necked round-bottom flask, was placed a solution of2-(4-bromo-3-fluorophenoxy)acetaldehyde (20 g, 91.74 mmol, 1.00 equiv)in MeOH (200 ml). To this was added 1-methylpiperazine (9.6 g, 95.90mmol, 1.05 equiv) followed by addition of HOAc (2 ml). The resultingsolution was allowed to react, with stirring, for 2 hours while thetemperature was maintained at 20° C. To the mixture was added NaBH₃CN(7.2 g, 110.77 mmol, 1.21 equiv), while the temperature was cooled to 0°C. The resulting solution was allowed to react, with stirring, for 1hour while the temperature was maintained at 0° C. The reaction progresswas monitored by TLC (CH₂Cl₂/MeOH=15:1). The mixture was concentrated byevaporation under vacuum using a rotary evaporator. The resultingsolution was extracted three times with 300 ml of EtOAc and the organiclayers combined. The resulting mixture was washed once with 500 ml ofbrine. The mixture was dried over Na₂SO₄. A filtration was performed.The filtrate was concentrated by evaporation under vacuum using a rotaryevaporator. The residue was purified by eluting through a column with a(EtOAc/PE 1:50˜ MeOH/DCM 1:30) solvent system. This resulted in 9 g(30%) of 1-(2-(4-bromo-3-fluorophenoxy)ethyl)-4-methylpiperazine as ayellow oil.

LC-MS: [M+H]⁺ calcd for C₁₃H₁₉BrFN₂O: 317, found: 317.

H-NMR: 1H NMR (400 MHz, d₆-DMSO) δ 2.30 (3H, s), 2.48-2.61 (7H, m),2.79-2.82 (2H, m), 4.05-4.08 (2H, m), 6.60-6.71 (2H, m), 7.37-7.71 (1H,m).

Step 7:

Into a 250 ml 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen, was placedN-(3-fluorobenzyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide(8 g, 21.67 mmol, 1.00 equiv). To this was added1-(2-(4-bromo-3-fluorophenoxy)ethyl)-4-methylpiperazine (7 g, 22.07mmol, 1.02 equiv) followed by addition of Na₂CO₃ (11.7 g, 110.39 mmol,5.09 equiv) and Pd(dppf)Cl₂ (0.8 g). To the mixture was added DMF (80ml). The resulting solution was allowed to react, with stirring,overnight while the temperature was maintained at 100° C. The reactionprogress was monitored by TLC (EtOAc). The reaction mixture was cooledto 15° C. in a bath of H₂O. The reaction mixture was then quenched byadding 400 ml of H₂O. The resulting solution was extracted three timeswith 100 ml of EtOAc and the organic layers combined and dried overanhydrous Na₂SO₄. A filtration was performed. The filtrate wasconcentrated by evaporation under vacuum using a rotary evaporator. Theresidue was purified by eluting through a silica gel column with a 30/1CH₂Cl₂/MeOH solvent system. This resulted in 6.4 g (62%) of compound 139as a yellow solid.

LC-MS: [M+H]⁺ calcd for C₂₈H₃₂F₂N₃O₂: 480, found: 480.

H-NMR: 1H NMR (400 MHz, d₆-DMSO) δ 2.18-2.72 (13H, d), 3.33 (2H, s),3.53 (2H, s), 4.08-4.13 (2H, m), 4.29-4.30 (2H, m), 6.87-7.44 (11H, m),8.65 (1H, s).

Step 8:

Into a 1000 ml 3-necked round-bottom flask, was placed EtOH (200 ml). Tothe above HCl (g) was purged in. This was followed by the addition of asolution of 139 (6.4 g, 13.33 mmol, 1.00 equiv) in EtOH (300 ml), whichwas added dropwise with stirring. The resulting solution was allowed tostir for 0.5 hour while the temperature was maintained at 10° C. Afiltration was performed and the filter cake was collected. Thisresulted in 6.8 g (92%) of 139 di-HCl salt as a white solid.

LC-MS: [M-2HCl+H]+ calcd for C₂₈H₃₂F₂N₃O₂: 480, found: 480.

H-NMR: 1H NMR (400 MHz, D₂O) δ 2.83 (3H, s), 3.49-3.78 (11H, m),4.29-4.30 (2H, s), 4.49 (2H, s), 6.96-7.10 (5H, m), 7.32-7.50 (6H, m),8.74 (1H, s).

Alternative Synthesis of KX2-402

Preparation of 4-(2-(4-bromophenoxy)ethyl)morpholine (2a)

A 5 L three-necked round-bottomed flask, equipped with mechanicalstirrer, thermometer with adapter, condenser, and nitrogen inlet (on topof condenser), was charged with 1a (140.7 g, 0.756 mol), 4-bromophenol(130.6 g, 0.755 mol), anhydrous K2CO₃ powder (367.6 g, 2.66 mol, 3.5eq.), and acetonitrile (1.3 L). The mixture was vigorously stirred(blade touching bottom of flask) at 80° C. (overnight), followed bydilution with DCM (500 mL) and heptane (200 mL) and filtration thruCelite. Evaporation to dryness (rotovap, then high vac) gave 2a as alight yellow oil (216.00 g, yield of 100%, 96.3% AUC, contains 3.7%unreacted bromophenol). This material was used successfully withoutfurther purification.

¹H NMR (CDCl₃) δ 2.57 (t, 4H), 2.79 (t, 2H), 3.73 (t, 4H), 4.08 (t, 2H),6.78 (d, 2H), 7.37 (d, 1H). MS (from LC/MS): m/z 287.1 [M+1].

Bromophenol can be readily removed was demonstrated on a 2 g sample byfirst dissolving it in toluene (8 g) and washing with 8 g of 15% aqueousNaOH; HPLC showed no trace of unreacted bromophenol in the recoveredproduct (1.97 g; 98.5% recovery).

Analytical Data for1-(2-(4-bromo-3-fluorophenoxy)ethyl)-4-ethylpiperazine (2d)

¹H NMR (CDCl₃) δ 1.10 (t, 3H), 2.42 (q, 2H), 2.51 (br s, 4H), 2.62 (brs, 4H), 2.81 (t, 2H), 4.07 (t, 2H), 6.61 (ddd, 1H), 6.70 (dd, 1H), 7.39(dd, 1H). MS (from LC/MS): m/z 332.0 [M+1].

Preparation of 2-fluoro-4-(2-morpholinoethoxy)phenylboronic Acid (8a)

To stirred and cooled (dry ice/MeOH bath) dry ether (5 mL) was added 4.4mL (8.8 mmol) of 2 M n-BuLi over a period of 1 min. After continuedstirring and cooling (10 min) under nitrogen, a solution of 2b (2.21 g,7.27 mmol) in dry ether (8 mL) was added drop wise over a period of 5min. The resulting white suspension was stirred and cooled (dry ice/MeOHbath) an additional 30 min followed by the addition of a solution oftriisopropyl borate (1.78 g, 9.45 mmol) in dry ether (2 mL) over aperiod of 3 min. The reaction mixture was stirred over the cold bath anadditional 35 min followed by warming to room temperature over a periodof 1.25 h. A solution of NaOH (2.5 g) in water (5 mL), then some morewater (5 mL) and the mixture stirred at RT (30 min). After discardingthe top organic layer, the bottom aqueous layer was washed with ether(2×10 mL), diluted with DCM (20 mL), and then, with stirring, treatedneutralized to pH 7 by the slow addition of concentrated HCl (6.2 mL).After setting aside the bottom organic layer, the aqueous layer wasextracted with DCM (20 mL). The combined organics were dried (Na₂SO₄),filtered, and evaporated to dryness (along with a heptane chase followedby high vacuum drying) to give the desired product as a white solid(1.90 g, 94.8% AUC, yield of 97%) which was used successfully withoutfurther purification.

¹H NMR (DMSO-d₆) δ 2.45 (br s, 4H; overlaps with DMSO), 2.7 (br s, 2H),3.6 (br s, 4H), 4.1 (br s, 2H), 6.6-6.8 (m, 2H), 7.50 & 7.68 (twotriplets, total 1H; from probably two different forms of the boronicacid). MS (from LC/MS; 94.8% AUC with RT of 0.8 min): m z 270.1 [M+1]

Analytical Data for2-fluoro-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenylboronic Acid (8b)

¹H NMR (CDCl₃) δ 2.32 (s, 3H), 2.54 (br s, 4H), 2.63 (br s, 4H), 2.80(br s, 2H), 4.1 (t, 2H; overlaps with EtOAc signal), 6.5-6.8 (m, 2H),7.7 (t, 1H). MS (from LC/MS): m/z 283.6 [M+1].

Preparation of 2-(4-bromophenyl)-N-(3-fluorobenzyl)acetamide (9a)

To a mixture of (4-bromophenyl)acetic acid (1.00 g, 4.65 g) and1,1′-carbonyldiimidazole in THF was added 3-fluorobenzylamine and themixture stirred at room temperature. The mixture was diluted with EtOAcand water. After shaking, the organic layer was washed with water, dried(Na₂SO₄), filtered, and evaporated to dryness. Crystallization(EtOAc/heptane) gave 0.85 g (yield of 57%) of 9a as a white crystallinesolid.

¹H NMR (CDCl₃) δ 3.59 (s, 2H), 4.40 (d, 2H), 5.7 (br s, 1H), 6.85-7.00(m, 3H), 7.16 (d, 2H), 7.22-7.32 (m, 1H), 7.48 (d, 2H). MS (from LC/MS):m/z 323.3 [M+1]

Preparation of ethyl4-(3-fluoro-4-(6-(2-(3-fluorobenzylamino)-2-oxoethyl)pyridin-3-yl)phenoxy)butanoate(KX2-409). To a mixture of 8c (704 mg, 1.5 mmol), 9c (323 mg, 1.0 mmol),and dimethoxyethane (4.2 g) was added a solution of K2CO₃ (412 mg, 3mmol) in water (1.5 mL). Immediately after then adding Pd(PPh₃)₄ (65 mg,0.056 mmol), the reaction mixture was deoxygenated by bubbling nitrogenthru the mixture and then it was stirred at 90° C. (oil bathtemperature) for ˜4 h. The small bottom aqueous layer was removed(pipette) and the mixture dried (Na₂SO₄), filtered, and evaporated todryness. Chromatography using 50-100% EtOAc in heptane gave several purefractions, which were concentrated to a volume of ˜4 mL followed bydilution with heptane (˜20 mL), resulting in crystallization of theproduct. Filtration, washing solid with heptane, and air drying gaveKX2-409 as a white crystalline solid (0.36 g, yield of 77%).

¹H NMR (DMSO-d₆) δ 1.19 (t, 3H), 2.00 (quintuplet, 2H), 2.48 (t, 2H;overlaps with DMSO), 3.74 (s, 2H), 4.08 (t, 2H), 4.09 (q, 2H), 4.32 (d,2H), 6.85-7.15 (m, 5H), 7.3-7.55 (m, 3H), 7.88 (ddd, 1H), 8.61 (s, 1H),8.68 (t, 1H). MS (from LC/MS): m/z 469.3 [M+1]

Analytical Data for2-(2′-fluoro-4′-(2-(4-methylpiperazin-1-yl)ethoxy)biphenyl-4-yl)-N-(3-fluorobenzyl)acetamide(KX2-402)

¹H NMR (CDCl₃) δ 2.30 (s, 3H), 2.49 (br s, 4H), 2.63 (br s, 4H), 2.84(t, 2H), 3.69 (s, 2H), 4.12 (t, 2H), 4.42 (d, 2H), 5.78 (br t, 1H), 6.72(dd, 1H), 6.77 (dd, 1H), 6.85-7.0 (m, 3H), 7.2-7.4 (m, 4H+CHCl₃),7.45-7.55 (m, 2H). MS (from LC/MS): m/z 480.6 [M+1]

Preparation of KX2-402, diHCl Salt

To a stirred suspension of KX2-402 (100 mg) in absolute EtOH (5 mL) wasadded 0.5 mL of 1.5 M HCl (in ethanol). The resulting homogeneoussolution was stirred at RT (20 min) and then evaporated to near dryness.The solid obtained was reconcentrated to dryness with added EtOH twiceto obtain 98 mg of white solid. (Yield=85.0%) ¹H NMR (D₂O): 2.89 (s,3H), 3.49-3.53 (m, 12H), 4.17-4.23 (m, 4H), 6.73-6.78 (m, 5H), 7.11-7.19(m, 6H).

¹H NMR (DMSO-d₆): 2.83 (s, 3H), 3.45-3.65 (m, 12H), 4.3 (s, 2H), 4.51(s, 2H), 6.92 (d, 1H), 7.03-7.06 (m, 4H), 7.36-7.36 (m, 4H), 7.45-7.46(m, 3H), 8.72 (s, 1H). MS (from LC/MS): m/z 480.3 [M+1 of free base].

Example 2: Cell Growth Inhibition

The drug concentration required to block net cell growth by 50% relativeto a control sample is measured as the GI₅₀. The GI₅₀s for several ofthe compounds of the invention were assayed as described herein.

The HT29 cell line is a NCI standard human colon carcinoma cell line.HT-29 cells were obtained from ATCC at passage 125 and were used forinhibition studies between passage 126-151. HT29 cells were routinelycultured in McCoy's 5A medium supplemented with Fetal Bovine Serum (1.5%v/v) and L-glutamine (2 mM).

The c-Src 3T3 is a mouse fibroblast NIH 3T3 normal cell line that hasbeen transfected with a point-mutant of human c-Src wherein tyrosine 527has been converted to a phenylalanine. This mutation results in“constitutively active” c-Src because phosphorylation on tyrosine 527results in auto-inhibition of Src by having it fold back on its own SH2domain. With a Phe there, this phosphorylation can't occur and thereforeauto-inhibition can't occur. Thus, the always fully active mutant Srcthen converts the normal mouse fibroblasts into rapidly growing tumorcells. Since the hyperactive Src is the main factor driving growth inthese cells (particularly when cultured under low growth serumconditions), compounds active in blocking this growth are thought towork by blocking Src signaling (e.g. as a direct Src kinase inhibitor oras an inhibitor acting somewhere else in the Src signaling cascade). Thecells were routinely cultured in DMEM supplemented with Fetal BovineSerum (2.0% v/V), L-glutamine (2 mM) and Sodium Pyruvate (1 mM).

In the BrdU Assay for cell growth inhibition, quantitation of cellproliferation was based on the measurement of BrdU incorporation duringDNA synthesis. The Cell Proliferation ELISA BrdU assay kit(colorimetric) was obtained from Roche Applied Science and performed asper vendor instructions.

Growth inhibition was expressed as a GI₅₀ where the GI₅₀ is the sampledose that inhibits 50% of cell growth. The growth inhibition (GI) isdetermined from the formula GI=(T₀−T_(n)×100/T₀−CON_(n)) where T₀ is theBrdU growth of untreated cells at time “0”, T_(n) is the BrdU growth oftreated cells at day “n” and CON_(n) is the control BrdU growth ofcontrol cells at day “n”. The GI₅₀ was extrapolated and the data plottedusing XL-Fit 4.0 software.

Actively growing cultures were trypsinized and cells were resuspended in190 μL of appropriate culture medium supplemented with 1.05% FBS in eachwell of a 96-well culture plate (1000 HT-29 cells; 2500 c-Src 3T3cells). For 96 well culture plate experiments, c-Src 3T3 medium wassupplemented with 10 mM HEPES buffer. HT-29 cells were seeded instandard tissue culture 96-well plates and c-Src 3T3 cells were seededin 96-well plates coated with Poly-D-lysine (BIOCOAT™). To increase CO₂diffusion, c-Src 3T3 96-well plates were incubated with their lidsraised by ˜2 mm using sterile rubber caps.

Seeded 96 well plates were allowed to attach overnight for 18-24 hours,either at 37° C. and 5% CO₂ for HT-29 or at 37° C. and 10% CO₂ for c-Src3T3. Approx 18-24 hours after seeding, the initial growth of cells (To)was determined for untreated cells using the BrdU assay. Samples werereconstituted in DMSO at 20 mM and intermediate dilutions made usingDMEM containing 10% FBS. The final assay concentrations were 1.5% forFBS and 0.05% for DMSO. Samples were added as 10 μL aliquots intriplicate and plates were incubated as above for ˜72 hours. Negative(vehicle) and positive controls (e.g., AZ (KX2-328)) were included.Plates were assayed for BrdU and the data analyzed as above for GI₅₀.

The results are shown in Table 7. In this table, the data is listed asGrowth % of Control, such that a lower number at an indicatedconcentration indicates a greater potency of the compound in blockinggrowth of that tumor cell line. All compounds were initially prepared as20 mM DMSO stock solutions and then diluted into buffer for the in vitrotumor growth assays. NG means no cell growth beyond the control and Tmeans the number of cells in the drug treated wells was less than in thecontrol (i.e. net cell loss). NT indicates that the test was notperformed. Compound AZ (KX2-328) is an ATP-competitive tyrosine kinaseinhibitor, as described in P16 et al., J. Med. Chem, 47:871-887 (2004).

As shown in Table 7, GI₅₀s were obtained for a number of the compoundsin other cell lines. These GI₅₀'s were determined using the standardtumor growth inhibition assays, similar to that described in detail forthe HT29 cell line above, and the following cell lines: colon tumor celllines KM12, lung cancer cell line H460 and lung cancer cell line A549(all are NCI standard tumor cell lines).

TABLE 7 HT-29 c-Src 3T3 Growth, % of Control Growth, % of Control Mean,n = 3 Mean, n = 3 KX-# CMPD 5 uM 500 nM 50 nM GI₅₀ 10 uM 1.0 uM 100 nMKX2-328 AZ T 10.0 73.0 99 nM (c-Src 3T3), 794 nM (HT29) T T 13.0 KX1-1361 T T 83.1 53 nM (c-Src 3T3), 484 nM (HT29) T T 46.3 105 nM (KM12) 280nM (H460) 330 nM (A549) KX1-305 2 T T 107.7 349 nM (c-Src 3T3), 877 nM(HT29), T T 35.0 410 nM (KM12) 890 nM (H460) 1.03 uM (A549) KX1-307 439.4 93.8 85.9 4.2 45.3 65.7 KX1-308 5 32.3 76.1 87.9 67.1 77.7 94.5KX1-312 9 33.7 67.6 93.7 12.1 94.5 98.5 KX1-306 3 T T 124.4 T T 47.0KX1-313 10 T T 80.2 T T 91.6 KX1-319 16 T T 101.2 T T 88.2 KX1-309 6 T T29.5 T T T KX1-310 7 T T 93.3 T T 101.8 KX1-311 8 T T 60.4 T T 81.3KX1-327 24 T T 31.6 >200 nM (c-Src 3T3), 680 nM (HT29) T T 81.3 KX1-31613 T 45.1 77.8 >200 nM (c-Src 3T3) T T 88.2 KX1-315 12 T 50.3 66.0 T88.1 89.3 KX1-314 11 14.4 83.7 53.21 39.3 88.4 93.6 KX1-317 14 T 64.083.5 T 85.6 94.2 KX1-318 15 T 93.2 164.7 T 71.0 91.4 KX1-320 17 86.2132.0 111.2 73.1 86.5 90.4 KX1-321 18 23.7 118.1 127.2 55.8 96.2 95.5KX1-322 19 T 87.2 114.1 3,730 nM (Src 3T3) T T 94.6 KX1-323 20 60.8106.9 105.6 93.2 97.3 96.6 KX1-324 21 NG 95.7 91.0 T 90.0 96.0 KX1-32522 T T 85.0 207 nM (c-Src 3T3), 215 nM (HT29) T 54.2 97.6 KX1-326 2343.7 73.2 65.4 55.7 87.3 92.2 KX1-329, 25 T T 101 269 nM (c-Src 3T3),338 nM (HT29) T T 96.0 KX1-357 26 NT NT NT 9.0 95.4 101.3 KX1-358 27 NTNT NT 82.7 91.4 92.2 KX2-359 28 T T T 34 nM (c-Src 3T3), 45 nM (HT29) TT T KX2-360 54 T T 91 T T 106.0 KX2-361 76 T T T 11 nM (c-Src 3T3), 10nM (HT29) T T T KX2-362 78 T T 86 56 nM (c-Src 3T3), 56 nM (HT29) T T101 KX2-363 79 T 67 92 100 70 92 KX2-364 82 T 80 105 T 81 92 KX2-365 40T T 88 133 nM (c-Src 3T3), 93 nM (HT29) T T 88 KX2-366 75 T 54 89 T 83103 KX2-367 41 T 6 64 T T 102 KX2-368, 29 T 70 107 27 101 99 slightlyinsoluble KX2-369 55 T 72 87 T 101 100 KX2-370 77 81 93 112 106 105 104KX2-371 81 16 33 98 16 72 75 KX2-372 80 T T T 58 nM (c-Src 3T3); 67 nM(HT-29) T T T KX2-373 72 T T 64 96 nM (c-Src 3T3); 639 nM (HT-29) T T 97KX2-374 115 T 57 74 T 84 110 KX2-375 36 T T 99 206 nM (c-Src 3T3); 354nM (HT-29) T T T KX2-376 74 T 93 96 >1,600 nM (c-Src 3T3); >400 nM(HT-29) T T T KX2-377 38 T T T 118 nM (c-Src 3T3); 111 nM (HT-29) T T TKX2-378 31 T 61 88 48 107 122 KX2-379 70 T 88 89 T 104 106 KX2-380 30 T50 100 T 119 124 KX2-381 33 T T 58 914 nM (c-Src 3T3); 375 nM (HT-29) TT 116 KX2-382 68 50 97 80 103 114 117 KX2-383 116 327 nM (c-Src 3T3);248 nM (HT-29) KX2-384 64 1,430 nM (c-Src 3T3); inactive(HT-29) KX2-38583 232 nM (c-Src 3T3) KX2-386 37 897 nM (c-Src 3T3); inactive (HT-29)KX2-387 38 inactive (c-Src 3T3); 1,860 nM (HT-29) KX2-388 66 >1,600 nM(c-Src 3T3); 906 nM (HT-29) KX2-389 60 Inactive (c-Src 3T3); inactive(HT-29) KX1-329 135 inactive (c-Src 3T3); inactive (HT-29) N-oxideKX2-390 114 797 nM (c-Src 3T3); 868 nM (HT-29) KX2-391 133 13 nM (c-Src3T3); 23 nM (HT-29) KX2-392 134 13 nM (c-Src 3T3); 21 nM (HT-29) KX2-393136 24 nM (c-Src 3T3); 52 nM (HT-29) KX2-394 137 13 nM (c-Src 3T3); 26nM (HT-29) NG = No growth, total growth inhibition; T = Cytotoxic Effecton Cells, negative growth; NT = Not tested

Table 7A below shows KXO1 inhibition of Src driven tumor cell growth incomparison to the ATP competitive Src inhibitors currently in clinicaltrials.

TABLE 7A c-Src527F/NIH3T3 HT29 (Colon) Compound GI₅₀ (nM) GI₅₀ (nM) KXO123 25 AZ28 87 647 Dasatinib 3 20 SKI-606 208 173 AZD0530 203 330

Table 7B shows KXO1 and KX2-361 inhibition in brain tumor cell lines.These GI50s were determined using standard tumor growth inhibitionassays, similar to those described in detail in this Example 7.

TABLE 7B GI50 of KX2-361, KXO1 and Dasatinib in brain tumor cell lines:Cell KX2-361 KX01 Dasatinib Line IC50 IC50 IC50 Organism DiseaseMorphology Tumorigenic Daoy 54.9 nM 13.6 nM 2927 nM Human DesmoplasticPolygonal Yes cerebellar medulloblastoma SK-N-MC 0.13 nM 5.8 nM 5114 nMHuman Neuroepithelioma Epithelial Yes SW1088 22.1 nM 76.1 nM 897.3 nMHuman Astrocytoma Fibroblast Yes LN-18 2.9 nM 14.5 nM 565.3 nM HumanGlioblastoma; glioma Epithelial Yes SK-N-FI 0.46 nM 1.7 nM 12.6 nM HumanNeuroblastoma Epithelial Yes U87 9.7 nM 33.1 nM 1586 nM HumanGlioblastoma; Epithelial Yes astrocytoma GL261 8.8 nM 13.7 nM 17.7 nMMouse Glioblastoma Epithelial Yes

Table 7C shows KXO1 and KX2-402 inhibition in renal tumor cell lines.These GI₅₀s were determined using standard tumor growth inhibitionassays, similar to those described in detail in this Example 7.

TABLE 7C GI50 of KX-2-402, KX01 and Dasatinib in renal tumor cell lines:Cell KX2-402 KXO1 Dasatinib Line GI50 GI50 GI50 Organism DiseaseMorphology Tumorigenic 769-P 8.4 nM 45.0 nM 46.3 nM Human Renal cellEpithelial Yes adenocarcinoma 786-O 57.7 nM 378.4 nM 2014 nM Human Renalcell Epithelial Yes adenocarcinoma Caki-2 5.0 nM 39.2 nM 14.2 nM HumanClear cell Epithelial Yes carcinoma ACHN 6.9 nM 33.2 nM 21.1 nM HumanRenal cell Epithelial Yes adenocarcinoma

Table 7D shows a summary of the results KXO1 inhibition in fivehepatocellular carcinoma cell lines. The table shows the IC₅₀s and IC₈₀sof KX2-391-MSA and Dasatinib in hepatocellular carcinoma cell lines(8.0×103 cells/wells, 1.5% FBS) @78 Hr; results from normalized responsedata:

TABLE 7D GI50 of KXO1 and Dasatinib in hepatocellular carcinoma celllines: KX2-391—MSA Dasatinib Cell Line IC₅₀ (nM) IC₅₀ (nM) IC₅₀ (nM)IC₅₀ (nM) HuH7  9  23 1972   7135 WRL-68 15  25 5650  45,580 PLC/PRF/513  24  15 >50,000 Hep 3B 26  88  86 >50,000 Hep G2 60 3658 NA NA

Samples of the test compounds were formulated in 100% DMSO to obtain 20mM stock solutions; stored @4° C. The IC₅₀s and IC₈₀s were determined asdescribed below. Huh7, WRL-68, PLC/PRF/5, Hep 3B, and Hep G2 humancancer lines were routinely cultured and maintained in a basal mediumcontaining 2% FBS @37° C., 5% CO₂. Cells were seeded @4.0×10^(3/190) pland 8.0×10^(3/190) pl per well of a 96-well plate. The assay medium wasbasal medium/1.5% FBS. Cells were cultured overnight in 96-well platesat 37° C., 5% CO₂ prior to KX2-391.MSA and Dasatinib addition. The testarticle dilutions were prepared as follows: 20 mM stock solution sampleswere diluted serially in basal medium/1.5% FBS using 1:3 dilutions,yielding 20× concentrations; 131 μM to 0.24 nM range. 10 μL of 20×dilutions were added to the appropriate wells (n=3) containing 190 μLcancer cell line; 6561 nM to 0.012 nM range of final concentrations.Vehicle control contained cells, no sample. Medium control containedcells, no sample, 0.03% DMSO (highest DMSO concentration present insamples). The treated cells were incubated for 72 hours at 37° C., 5%CO₂. On day 3, 10 μL MTT (5 mg/mL) were added to each well. Cells wereincubated in the presence of MTT for 4 hours @37° C., 5% CO₂. 90 μL 10%SDS(+HCl) was added to each well to lyse cells and solubilize formazan.Cells were then incubated overnight @37°, 5% CO₂. OD₅₇₀ measurementswere taken using BioTek Synergy HT multiplatform microplate reader.Growth inhibition curves IC₅₀s and IC₈₀s were determined using GraphPadPrism 5 statistical software.

Example 3: Inhibition of Isolated Kinases

It is believed that the conformation of Src outside cells vs. insidecells is markedly different, because inside cells, Src is embedded inmultiprotein signaling complexes. Thus, because the peptide substratebinding site is not well formed in isolated Src (as shown by Src x-raystructures), it is believed that the activity against isolated Src for apeptide substrate binding inhibitor would be weak. Binding to this sitewill require the inhibitor to capture the very small percentage of totalSrc protein in the isolated enzyme assay that is in the sameconformation that exists inside cells. This requires a large excess ofthe inhibitor to drain significant amounts of the enzyme from thecatalytic cycle in the assay.

However, inside cells this large inhibitor excess is not needed becausethe SH2 & SH3 domain binding proteins have already shifted the Srcconformation so that the peptide substrate binding site is fully formed.Now, low concentrations of the inhibitor can remove the enzyme from thecatalytic cycle since all of the enzyme is in the tight bindingconformation.

KX2-328 is AstraZeneca's published ATP-competitive Src inhibitor (AZ28)and is used as a positive control in many of the experiments describedherein. Note that KX2-391 has weak activity against isolated kinasesbecause the peptide binding site is not well formed outside of cells (aclose analog, KX2-394 is a little more potent against isolated Src), buthave very potent activity inside whole cells. Without wishing to bebound by theory, it is thought that the difference in activity isattributed to the fact that the peptide binding site is now fully formedin cells due to the allosteric effects of the binding protein partnersin the multi-protein signaling complexes, relative to isolated kinaseassays.

Table 8 illustrates percent activity of isolated kinases in the presenceof the AstraZeneca ATP-competitive inhibitor (KX2-328, AZ-28) or KX2-391relative to control (untreated) isolated kinases.

TABLE 8 Target AZ28 @ 10 μM KX2-391 @ 10 μM Abl(h) 1 120 CHK1(h) NT 105EGFR(h) 3 134 FGFR2(h) 94 94 Fyn(h) 2 85 IGF-1R(h) 110 101 IR(h) 125 112Lck(h) 1 109 Lyn(h) 0 113 MAPK2(h) 105 112 PDGFRβ(h) 98 110 PKCα(h) 111111 Pyk2(h) 43 97 Yes(h) 1 92 ZAP-70(h) 97 108 PI3 kinase 99 100

The AstraZeneca ATP competitive inhibitor shows the typical off targetkinase inhibition activity for ATP-competitive inhibitors, poorselectivity as evidenced by strong inhibition of Abl, EGFRTK, Fyn, Lck,Lyn & Yes. In contrast, poor inhibition of these off-target kinases isseen with KX2-391.

However, KX2-391 is a more potent inhibitor of Src-driven cell growth,assayed as described in Example 2. In the c-Src/NIH-3T3 engineered cellline, the GI₅₀ for AZ28 is 99 nM, vs. 13 nm for KX2-391, and in the NCIhuman colon cancer cell line HT29, the GI₅₀ for AZ28 is 794 nM, vs. 23nm for KX2-391. Similar to KX2-391, the GI₅₀ for KX2-394 in thec-Src/NIH-3T3 engineered cell line is 13 nM, and in the NCI human coloncancer cell line HT29, the GI₅₀ for KX2-394 is 794 nM, vs. 33 nm.

In separate examples, titration data indicate that AZ28 is a potentinhibitor of isolated Src (IC50=8 nM). The titration data with FAK showsthat AZ28 is at least ca. 100× less potent against isolated FAK(IC50>500 nM). Whereas, titration data indicate that KX2-391 and KX2-394are less potent inhibitors of isolated Src (IC50=46 μM and 5 μM,respectively). The titration data with FAK shows that KX2-391 andKX2-394 are similarly potent against isolated FAK (IC50>48 μM).

Note that AZ28 is 10-100× less potent against cell growth than againstisolated Src. This is typical of ATP competitive inhibitors since theconcentration of competing ATP is much higher in whole cells than in theisolated enzyme assays.

The compounds FIG. 43 shows KXO1 activity against isolated c-Src andFAK. KXO1 exhibited an IC₅₀=46 mM against cSrc.

Example 4: Effect of Compounds on Intracellular Phosphorylation Levels

HT29 (colon cancer) and c-Src527F/NIH-3T3 (Src transformed) cell lineswere treated with KX2-391 or with AstraZeneca's ATP competitive Srcinhibitor AZ28. AZ28 serves as a positive comparator to show what avalidated Src inhibitor should do in these assays. After treatment withcompound, cells were lysed, subjected to PAGE and probed with a batteryof antibodies. The antibodies were selected to determine whethercompounds caused changes in phosphorylation of known Src substrates. Inaddition, off-target protein phosphorylation was also investigated.Further, induction of apoptosis was evaluated via Caspase 3 cleavage.Multiple doses of each compound were tested because the trends inresponse to increasing drug concentration are the most reliableindicator of activity.

A dose response curve for KX2-391 was generated using the GI₅₀ for thiscompound in each of the two cell lines as the 1×concentration. Threeadditional doses 0.2×, 5× & 25× multiples the GI₅₀'s were also tested inaddition to a no drug control “C”. The same range of multiples of theGI₅₀ for AZ28 in these two cell lines was run as a comparison. As shownin FIG. 1, the expected dose response for Src-Y416 autophosphorylationwas obtained in both cell lines, and for both compounds. This dataindicates that KX2-391 is a Src inhibitor inside cells.

FIG. 2 shows phosphorylation of FAK Tyr 925, a known Srctransphorylation substrate within cells. KX2-391 and AZ28 inhibited Srctransphosphorylation. This data indicates that KX2-391 is a Srcinhibitor inside cells.

FIG. 3 shows phosphorylation of Shc Y239/240, a known Srctransphorylation substrate within cells. KX2-391 and AZ28 inhibited Srctransphosphorylation. This data indicates that KX2-391 is a Srcinhibitor inside cells.

FIG. 4 shows phosphorylation of Paxillin Y-31, a known Srctransphorylation substrate within cells. KX2-391 and AZ28 inhibited Srctransphosphorylation. This data indicates that KX2-391 is a Srcinhibitor inside cells. Note: paxillin Y-31 was not detected in HT29cells with or without added drug.

Cleavage of Caspase-3 is a good measure of induction of apoptosis. It isknown that AZ28 is not effective in inducing apoptosis in HT29 (coloncancer) and c-Src527F/NIH-3T3 (Src transformed) cell lines. In contrast,as shown in FIG. 5, KX2-391 is very effective in inducing apoptosis.

Since Src activity is very high in both HT29 (colon cancer) andc-Src527F/NIH-3T3 (Src transformed) cell lines, one would expect to seea reduction in the total phosphotyrosine levels when Src activity isinhibited. FIG. 6 indicates that this is true for both AZ28 and KX2-391.This data indicates that KX2-391 is a Src inhibitor inside cells.

PDGF receptor tyrosine kinase autophosphorylates on Y572/574. This isthought not to be a direct Src substrate in cells. It is known that AZ28is not a potent inhibitor of isolated PDGF receptor tyrosine kinase (seeTable 8). Nevertheless, a dose response reduction in PDGF receptorautophosphorylation is seen with AZ28, as shown in FIG. 7. This suggeststhat this is an indirect effect. Some effect is seen with KX2-391,however it is somewhat less potent. Thus, KX2-391 is less active thanAZ28 against indirect PDGF autophoshorylation inhibition. PDGF receptortyrosine kinase Y572/574 was not detected in HT29 cells with no drugadded (as well as with drug added).

FAK Y397 is mainly a FAK autophosphorylation site and only a poor Srctransphorylation site. AZ28 is not a potent FAK inhibitor (see isolatedenzyme data in Table 8). Nevertheless, some inhibition of FAKautophosphorylation in c-Src527F/NIH3T3 cells with AZ28 is shown in FIG.8. However, no inhibition of FAK autophosphorylation in c-Src527F/NIH3T3cells is seen with KX-391. The opposite is true in the NCI human coloncancer cell line HT29.

The isolated enzyme data shown in Table 8 demonstrated that AZ28 is apotent EGFR tyrosine kinase inhibitor. In agreement with this the tumorcell data in FIG. 9 shows that AZ28 potently inhibits EGFR tyrosinekinase autophosphorylation. This site is not a direct Srcphosphorylation site. The tumor cell data in FIG. 9 also shows thatKX-391 is less active against the off target autophosphorylation ofEGFRTK.

The inhibition of autophosphorylation correlates with the GI₅₀'s ofcompounds of the invention. FIGS. 44A and 44B show inhibition of Srcautophophorylation (Y416) by KXO1 as compared to Az28 inc-Src527F/NIH-3T3 cells and in HT-29 cells. The inhibition oftransphosphorylation also correlates with the GI₅₀'s of compounds of theinvention. FIGS. 44D and 44E show inhibition of Src transphosphorylationof She Y239/240 by KXO1 as compared to Az28 in c-Src527F/NIH-3T3 cellsand in HT-29 cells.

The compounds of the present invention show very high protein tyrosinekinase selectivity in whole cell assays. For example, FIG. 45 shows KXO1selectivity for protein tyrosine kinases in comparison to Dasatinib.

Example 5: Protection Against Noise-Induced Hearing Loss Using PTKInhibitors

Chinchillas (N=6) were used in studies of noise-induced hearing loss.The animals' hearing sensitivity was measured using standardelectrophysical techniques before the experimental manipulation. Inparticular, hearing thresholds were measured through evoked potentialsfrom recording electrodes chronically implanted in the inferiorcolliculus, following standard laboratory procedures. Animals wereanesthetized, the auditory bullae were opened, and the left and rightcochleas were visualized. The round window leading to the scala tympaniof the cochlea was used as the access point for drug application.Animals were treated with KX1-004, KX1-141, KX1-329 or KX2-328 (anon-ATP competitive inhibitor from AstraZeneca, KX2-238), emulsified inDMSO, in 1000 mM of saline solution, which was placed on the roundwindow of one ear.

A control solution of 3 mM DMSO in 1000 mM of saline solution was placedon the round window of the other ear. The solution was allowed to set onthe round window for 30 minutes, then the auditory bullae were closed.Subsequently, the animals were exposed to 4 kHz band noise at 105 dB SPLfor four hours. Following the noise exposure, the animals' hearing wastested at day 1, day 7, and day 21 to determine evoked potentialthreshold shifts. Permanent threshold shift was assessed at day 21.

FIGS. 10-12 show the average threshold shifts for animals treated withKX1-004, KX1-141, KX1-329 or KX2-328. In particular, FIG. 10 showsaverage threshold shifts after exposure to 0.5 kHz, 1 kHz, 2 kHz, 4 kHz,and 8 kHz band noise on day 1 after experimental manipulation. FIG. 11shows average threshold shifts after exposure to 0.5 kHz, 1 kHz, 2 kHz,4 kHz, and 8 kHz band noise on day 7 after experimental manipulation.FIG. 12 shows average threshold shifts after exposure to 0.5 kHz, 1 kHz,2 kHz, 4 kHz, and 8 kHz band noise on day 21 after experimentalmanipulation. As shown in FIGS. 10-12, in most cases, the average dBthreshold shifts for ears treated with KX1-004, KX1-141, KX1-329 orKX2-328 were lower, indicating that the compounds reduced the level ofhearing loss in treated animals relative to the untreated controlanimals.

Example 6: Protection Against Cisplatin-Induced Hearing Loss Using PTKInhibitors

The effects of high level noise and ototoxic drugs, such as cisplatin orthe class of aminoglycosides, share several common features in the innerear. First, the noise and/or drugs alter the free radical/antioxidantlevels in the cochlea (inner ear). The increase in free radicals hasbeen shown to be a causative factor in the apoptotic death of thesensory cells. Guinea pigs (N=7) were used in studies ofcisplatin-induced hearing loss. The animals' hearing sensitivity wasmeasured using standard electrophysical techniques before theexperimental manipulation. In particular, hearing thresholds weremeasured through evoked potentials from recording electrodes chronicallyimplanted in the inferior colliculus, following standard laboratoryprocedures. Animals were anesthetized and treated with cisplatin.Subsequently, the animals' hearing was tested to determine evokedpotential threshold shifts.

FIG. 13 shows threshold shifts for a number of guinea pigs afterexposure to 2 kHz, 4 kHz, 8 kHz, 12 kHz, 16 kHz and 20 kHz band noiseafter treatment with cisplatin. FIG. 14 shows the threshold shifts foranimals treated with KX1-004. KX1 is CH65. Animals were treatedsubcutaneously with KX1-004 prior to the cisplatin-induced hearing loss.FIG. 15 shows the median CAP thresholds after cisplatin-induced hearingloss for both the untreated control animals and the KX1-004(CH65)-treated animals. As shown in FIG. 15, KX1-004 treatment protectedagainst ciplatin-induced hearing loss.

Example 7: Effect of Compounds on Osteoclast Formation

To determine the effect of the compounds on osteoclast formation, thecompounds were added to osteoclast precursors derived from spleen cells.For the generation of spleen-derived osteoclasts, spleen cellscomprising osteoclast precursors were treated with Rapamycin, KX1-141,KX2-328 (AstraZeneca compound), or KX1-329 for 5 days in the presence ofreceptor activator of nuclear factor-κB ligand (RANKL) and macrophagecolony-stimulating factor (M-CSF). In in vitro murine or humanosteoclast models, soluble RANKL enables osteoclast precursors todifferentiate in the presence of M-CSF (Quinn, et al.; 1998,Endocrinology, 139, 4424-4427; Jimi, et al.; 1999, J Immunol., 163,434-442). The untreated control cells were incubated in the presence ofRANKL and M-CSF alone. Rapamycin was used as a positive control for theinhibition of osteoclast formation. FIG. 16 shows that increasingconcentrations of Rapamycin (0.0001 μM, 0.001 μM, 0.01 μM, or 0.1 μM),KX1-141 (0.5 μM, 2.5 μM, 12.5 μM, or 20 μM), KX2-328 (0.02 μM, 0.1 μM,0.5 μM, or 2.5 μM), or KX1-329 (0.06 μM, 0.3 μM, 1.5 μM or 7.5 μM) wereadded to the spleen cells. The cells were stained as shown in FIG. 16.All four compounds, including the positive control Rapamycin, inhibitedthe formation of osteoclasts compared to the untreated control.

For generating spleen-derived osteoclasts, spleen cells were treated asdescribed above. FIG. 17 shows that increasing concentrations ofRapamycin (0.1 nM, 1 nM, 10 nM, or 100 nM), KX1-141 (0.5 μM, 2.5 μM,12.5 μM, or 20 μM), KX2-328 (0.02 μM, 0.1 μM, 0.5 μM, or 2.5 μM), orKX1-329 (0.06 μM, 0.3 μM, 1.5 μM or 7.5 μM) were added to the spleencells. Cells were then stained with the osteoclast marker,tartrate-resistant acid phosphatase (TRAP) to visualize differentiatedcells. The numbers of TRAP-positive osteoclasts were counted. All fourcompounds, including the positive control Rapamycin, reduced the numberof TRAP-positive osteoclasts compared to the untreated control (Ctr).

Example 8: Effect of Compounds on Osteoclast Survival

To determine the effect of the compounds on osteoclast survival,osteoclasts were treated with Rapamycin, KX1-141, KX2-328, or KX1-329for 48 hours in the presence of RANKL and M-CSF. The untreated, controlcells were incubated in the presence of RANKL and M-CSF alone. Rapamycinwas used as a positive control for the inhibition of osteoclastsurvival. FIG. 18 shows that increasing concentrations of Rapamycin(0.001 μM, 0.01 μM, 0.1 μM, or 1 μM), KX1-141 (0.5 μM, 2.5 μM, 12.5 μM,or 20 μM), KX2-328 (0.02 μM, 0.1 μM, 0.5 μM, or 2.5 μM), or KX1-329(0.06 μM, 0.3 μM, 1.5 μM or 7.5 μM) were added to the osteoclasts. Thecells were stained as shown in FIG. 18. All four compounds, includingthe positive control Rapamycin, inhibited the survival of osteoclastscompared to the untreated control.

As described above, osteoclasts were treated with Rapamycin, KX1-141,KX2-328, or KX1-329 for 48 hours in the presence of RANKL and M-CSF.FIG. 19 shows that increasing concentrations of Rapamycin (0.1 nM, 1 nM,10 nM, or 100 nM), KX1-141 (0.5 μM, 2.5 μM, 12.5 μM, or 20 μM), KX2-328(0.02 μM, 0.1 μM, 0.5 μM, or 2.5 μM), or KX1-329 (0.06 μM, 0.3 μM, 1.5μM or 7.5 μM) were added to the osteoclasts. Cells were then stainedwith TRAP and the number of TRAP-positive osteoclasts were counted. Allfour compounds, including the positive control Rapamycin, reduced thenumber of TRAP-positive osteoclasts compared to the untreated control.

Example 9: Effect of Compounds on Bone Resorption In Vitro

To determine the effects of the compounds on osteoclast formation onbone slices, the bone slices were treated with Rapamycin, KX1-141,KX2-328, or KX1-329. FIG. 20A shows that increasing concentrations ofRapamycin (0.1 nM, 1 nM, or 10 nM), KX1-141 (2.5 μM, 12.5 μM, or 20 μM),KX2-328 (0.1 μM, 0.5 μM, or 2.5 μM), or KX1-329 (0.3 μM, 1.5 μM or 7.5μM) were added to the bone slices. The number of osteoclasts on the boneslices were counted. All four compounds, including the positive controlRapamycin, reduced the number of osteoclasts on the bone slices comparedto the untreated control (Ctr).

During the resorption of bone, osteoclasts form resorption pits. Todetermine the effects of the compounds on resorption pit formation onbone slices, the bone slices were treated with Rapamycin, KX1-141,KX2-328, or KX1-329, as described above. FIG. 20B shows that increasingconcentrations of Rapamycin (0.1 nM, 1 nM, or 10 nM), KX1-141 (2.5 μM,12.5 μM, or 20 μM), KX2-328 (0.1 μM, 0.5 μM, or 2.5 μM), or KX1-329 (0.3μM, 1.5 μM or 7.5 μM) were added to the bone slices. The number ofresorption pits on the bone slices was determined. The compounds reducedthe number of resorption pits on the bone slices compared to theuntreated control (Ctr).

Bone slices were treated as indicated above. FIG. 21A shows thatincreasing concentrations of Rapamycin (0.001 μM, 0.01 μM, or 0.1 μM),KX1-141 (2.5 μM, 12.5 μM, or 20 μM), KX2-328 (0.1 μM, 0.5 μM, or 2.5μM), or KX1-329 (0.3 μM, 1.5 μM or 7.5 μM) were added to the boneslices. The bone slices were then stained with TRAP. All four compounds,including the positive control Rapamycin, reduced the number ofTRAP-positive osteoclasts on the bone slices compared to the untreatedcontrol. Notably, 12.5 μM KX1-141 significantly reduced the number ofTRAP-positive osteoclasts on the bone slices compared to the untreatedcontrol.

Bone slices were treated as indicated above. FIG. 21B shows thatincreasing concentrations of Rapamycin (0.001 μM, 0.01 μM, or 0.1 μM),KX1-141 (2.5 μM, 12.5 μM, or 20 μM), KX2-328 (0.1 μM, 0.5 μM, or 2.5μM), or KX1-329 (0.3 μM, 1.5 μM or 7.5 μM) were added to the boneslices. The bone slices were stained with Toluidine Blue to revealresorption pits, which are indicators of osteoclast-mediated resorptionof bone. All four compounds, including the positive control Rapamycin,reduced the number of resorption pits on the bone slices compared to theuntreated control.

Example 10: Effect of Compounds on Osteoblasts

The enzyme alkaline phosphatase has been used as an indicator ofosteoblast activity, as it is involved in making phosphate available forcalcification of bone. To determine the effects of the compounds onosteoblast activity, osteoblasts were treated with KX1-141 (0.5 μM, 2.5μM, 12.5 μM, or 20 μM), KX2-328 (0.02 μM, 0.1 μM, 0.5 μM, or 2.5 μM), orKX1-329 (0.06 μM, 0.3 μM, 1.5 μM or 7.5 μM) and alkaline phosphataseexpression was determined (nM alkaline phosphatase/μg protein/min (FIG.22). As controls, osteoblasts were treated with medium alone, dimethylsulfoxide (DMSO), or bone morphogenic protein-2 (BMP2). BMPs, defined asosteoinductive by their ability to induce osteogenesis when implanted inextraskeletal sites, are thought to mediate the transformation ofundifferentiated mesenchymal cells into bone-producing osteoblasts.

To determine the effects of the compounds on osteoblast activity andprotein expression, osteoblasts were treated with medium, DMSO, BMP2,KX1-141, KX2-328, or KX1-329 as indicated above. The proteinconcentration in cell lysates was determined (μg/10pl) (FIG. 23).Notably, KX1-141 increased protein concentration when administered at0.5 μM and 2.5 μM, but reduced protein concentration in cell lysateswhen administered at 12.5 μM and 20 μM. Additionally, KX1-329 increasedprotein concentration when administered at 0.06 μM and 0.3 μM, butreduced protein concentration when administered at 1.5 μM and 7.5 μM.

Example 11: Effect of Compounds on Obesity

The following example illustrates that the compounds of the presentinvention could be used to treat obesity. The compounds are tested usinga method described previously (Minet-Ringuet, et al.; 2006,Psychopharmacology, Epub ahead of print, incorporated herein byreference). Thirty male Sprague-Dawley rats initially weighing 175-200 gare housed in individual Plexiglas cages with an artificial 12:12-hlight-dark cycle (lights on at 08:00 h) in a room maintained at 24-1° C.and 55±5% humidity. Food and water are available ad libitum throughout.All rats are fed with a medium fat diet (metabolizable energy 17.50kJ/g) composed of 140 g/kg of whole milk protein, 538.1 g/kg ofcornstarch, 87.6 g/kg of sucrose, and 137 g/kg of soya bean oil, andthis diet is supplemented with minerals and vitamins (mineral salts 35g/kg, vitamins 10 g/kg, cellulose 50 g/kg, and choline 2.3 g/kg). Thisfood, named P14-L, which resembles the usual human diet (14% proteins,31% lipids, and 54% carbohydrates) is prepared in the laboratory in theform of a powder.

Several doses of the compound of the instant invention are tested: 0.01,0.1, 0.5, and 2 mg/kg, in addition to the control solution. The compoundis solubilized in water and then incorporated into the diet. The basalfood intake is recorded during the adaptation period and used todetermine the daily quantity of the compound of the instant inventionincorporated into food. The compound is mixed into the food in thelaboratory. After 1 week of adaptation to the laboratory conditions, therats are separated into five groups (n==6 per group) with homogenousweight and receive the compound of the instant invention in their foodfor 6 weeks. Weight is recorded three times per week. Body compositionis measured at the end of the study by dissection and by weighing themain organs and tissues. Briefly, rats are deeply anesthetized by anintraperitoneal injection of an overdose of anesthetic (sodiumpentobarbital 48 mg/kg) and heparinized (100 U heparin/100 g bodyweight). They are bled (to avoid coagulation in tissues) by sectioningthe vena cava and abdominal aorta before removal and weighing of themain fresh organs (liver, spleen, kidneys, and pancreas) and tissues(perineal and scapular brown adipose tissue. epididymal,retroperitoneal, visceral, and subcutaneous white adipose tissues(WATs). and carcass defined by muscles and skeleton). The compounds ofthe invention, which reduce the body weight of the animals, may be usedto treat obesity in a subject.

Example 12: Effect of Compounds on Insulin-Induced GLUT4 Translocationin 3T3-L1 Adipocytes

The following example illustrates that the compounds of the presentinvention could be used to treat diabetes. The compounds are testedusing a method described previously (Nakashima, et al.; 2000, J. Biol.Chem., 275, 12889-12895). Either control IgG, or the compound of theinstant invention is injected into the nucleus of differentiated 3T3-L1adipocytes on coverslips. Glutathione S-transferase fusion proteins areeach coinjected with 5 mg/ml sheep IgG for detection purposes. Prior tostaining, the cells are allowed to recover for a period of 1 h. Cellsare starved for 2 hr in serum-free medium, stimulated with or withoutinsulin (0.5 nM or 17 nM) for 20 min and fixed.

Immunostaining is performed using rabbit polyclonal anti-GLUT4 (F349) (1μg/ml). Each fluorescein isothiocyanate-positive microinjected cell isevaluated for the presence of plasma membrane-associated GLUT4 staining.Control cells are injected with preimmune sheep IgG and then processedin the same way as experimentally injected cells. As quantitated byimmunofluorescent GLUT4 staining, insulin leads to an increase in GLUT4translocation to the plasma membrane. Cells are incubated withwortmannin as a control to block basal and insulin-induced GLUT4translocation. The compounds of the instant invention could stimulateinsulin-induced GLUT4 translocation, which could indicate thatadministration of the compounds of the invention inhibited kinaseactivity, e.g., PTEN function, resulting in an increase in intracellularphosphatidylinositol 3,4,5-triphosphate levels, which stimulates GLUT4translocation.

Example 13: Effect of Compounds on Retinal Neovascularization

The following example illustrates that the compounds of the presentinvention could be used to treat eye diseases, e.g., maculardegeneration, retinopathy and macular edema. The effect of compounds onretinal neovascularization is determined using a model of retinalneovascularization as previously described (Aiello, et al.; 1995, Proc.Natl. Acad. Sci., 92, 10457-10461). Briefly, C57Bl/6J mice are exposedto 75% O₂ from postnatal day 7 (P7) to P12 along with nursing mothers.At P12, the mice are returned to room air. Intraocular injections areperformed at P12 and sometimes P14 as described below. At P17 the miceare sacrificed by cardiac perfusion of 4% paraformaldehyde inphosphate-buffered saline and the eyes are enucleated and fixed in 4%paraformaldehye overnight at 4° C. before paraffin embedding.

Mice are deeply anesthetized with tribromoethanol for all procedures.The lid fissure is opened (e.g., using a no. 11 scalpel blade) and theeye is proptosed. Intravitreal injections are performed by firstentering the left eye with an Ethicon TG140-8 suture needle at theposterior limbus. A 32-gauge Hamilton needle and syringe are used todeliver the compound of the instant invention diluted in Alcon balancedsalt solution through the existing entrance site. The eye is thenrepositioned and the lids are approximated over the cornea. Repeatinjections are performed through a previously unmanipulated section oflimbus 2 days later. As a control, equal amounts of saline are injectedto the right eye.

Over 50 serial 6-μm paraffin-embedded axial sections are obtainedstarting at the optic nerve head. After staining with periodicacid/Schiff reagent and hematoxylin (Pierce, et al.; 1995, Proc. Natl.Acad. Sci. USA., 92, 905-909; Smith et al.; 1994, Invest. Ophthal. Vis.Sci., 35, 101-111), 10 intact sections of equal length, each 30 μmapart, are evaluated for a span of 300 μm. Eyes exhibiting retinaldetachment or endophthalmitis are excluded from evaluation. All retinalvascular cell nuclei anterior to the internal limiting membrane arecounted in each section by a fully masked protocol. The mean of all 10counted sections yield average neovascular cell nuclei per 6-μm sectionper eye. No vascular cell nuclei anterior to the internal limitingmembrane are observed in normal, unmanipulated animals (Smith et al.;1994, Invest. Ophthal. Vis. Sci., 35, 101-111). Reducedneovascularization could be observed in the eyes treated with thecompounds of the instant invention as compared to the eyes in the salinecontrol group.

Example 14: Identification of Compounds that Modulate Kinase SignalingCascade Associated with Stroke

Many animal models for stroke have been developed and characterized, seee.g., Andaluz, et al., Neurosurg. Clin. North Am., vol. 13:385-393(2002); Ashwal, S. and W. J. Pearce., Curr. Opin. Pediatr., vol13:506-516 (2001); De Lecinana, et al., Cerebrovasc. Dis., vol.11(Suppl. 1):20-30 (2001); Ginsberg and Busto, Stroke, vol. 20:1627-1642(1989); Lin, et al., J. Neurosci. Methods, vol. 123:89-97 (2003);Macrae, I. M., Br. J. Clin. Pharmacol., vol. 34:302-308 (1992); McAuley,M. A., Cerebrovasc. Brain Metab. Rev., vol. 7:153-180 (1995); Megyesi,et al., Neurosurgery, vol. 46:448-460 (2000); Stefanovich, V. (ed.).,Stroke: animal models. Pergamon Press, Oxford (1983); and Traystman, R.J., ILAR J. 44:85-95 (2003), each of which is hereby incorporated byreference in its entirety. For a review of animal models of focal(stroke) and global (cardiac arrest) cerebral ischemia, see e.g.,Traystman, ILAR J., vol. 44(2):85-95 (2003) and Carmichael, NeuroRx®:The Journal of the American Society for Experimental NeuroTherapeutics,vol. 2:396-409 (2005, each of which is hereby incorporated by referencein its entirety.

Compounds that modulate cell death in stroke are identified using any ofthe art-recognized models for stroke. In the studies described herein,intra-arterial suture occlusion of the middle cerebral artery (MCA), aprocedure known as MCAo, through the internal carotid artery is used asa model for cell death in stroke. In the control and test group of rats,the external carotid artery is transected, the common carotid artery istied off, and the external carotid artery is then used as a pathway topass a suture through the internal carotid artery, wherein the suturelodges in the junction of the anterior and middle cerebral arteries. Toreduce subarachnoid hemorrhage and premature reperfusion, the suture ispreferably coated with an agent such as silicone. The suture is used toocclude the MCA, e.g., for a duration of 60, 90, or 120 minutes and topermanently occlude the MCA.

In the test group, rats are administered a compound of the invention ata variety of times prior to, during and after occlusion of the MCA withthe suture. The effects of the compound on the test group is compared tothe effects observed in the control group, for example, by measuring theextent of cell death in each MCAo group. Typically, in the controlgroup, the pattern of cell death follows a progression from earlyinfarction in the striatum to delayed infarction in the dorsolateralcortex overlying the striatum. Striatal is mostly necrotic and occursrapidly. The pattern of cell-death in the test group is compared to thatof the control group to identify compounds that modulate cell death instroke.

Example 15: Identification of Compounds that Modulate Kinase SignalingCascade Associated with Atherosclerosis

Many animal models for atherosclerosis have been developed andcharacterized. For a review of animal models of atherosclerosis,restenosis and endovascular graft research, see e.g., Narayanaswamy etal., JVIR, vol. 11(1): 5-17 (2000), which is hereby incorporated byreference in its entirety. Atherosclerosis is induced in a suitableanimal model using a high fat/high cholesterol (HFHC) diet. The testanimal is an animal that contains cholesterol ester transferase, such asthe rabbit or the swine. The HFHC diet is produced, e.g., usingcommercial chow supplemented with fat. Cholesterol intake is between0.5-2.0% of the diet. A test group of animals, e.g., rabbits or swine,receives a compound of the invention. The effect of the test compound iscompared to the effects of atherosclerosis in the untreated, controlgroup of animals. Effects that are compared include, for example, thedegree of plaque formation, the number and/or frequency of myocardialinfarctions observed in each group of animals, and the extent of tissuedamage secondary to myocardial infarction exhibited in coronary tissue.

Myocardial infarction is studied using a variety of animal models suchas rats and mice. The majority of myocardial infarctions result fromacute transbotic occlusion of pre-existing atherosclerotic plaques ofcoronary arteries, which is mimicked in animal models by ligation of theleft coronary artery in e.g., rats and mice. Myocardial infarctioninduces global changes in the ventricular architecture, a process calledventricular remodeling. The infarcted heart progressively dilates andaccelerates the deterioration of ventricular dysfunction that eventuallyresults in heart failure.

Myocardial ischemia is induced in test and control groups of animals,e.g., mice or rats, by ligating the left anterior descending coronaryartery. The affected heart tissue is contacted with a compound of theinvention, for example, by intraperitoneal (i.p.) injections, after theinduction of ischemia. High resolution magnetic resonance imaging (MRI),dry weight measurements, infarct size, heart volume, and area at riskare determined 24 hours postoperatively. Survival rates andechocardiography are determined at various times postoperatively in therats receiving injections of the compound of the invention. Othereffects of the test compound are compared to the control group of rats.For example, changes in left ventricular geometry and function arecharacterized using echocardiography to compare end-diastolic diameters,relative wall thickness, and the percentage of fractional shortening. Inexcised hearts, the infarct size calculated and expressed as apercentage of left ventricular surface area.

Example 16: Identification of Compounds that Modulate Kinase SignalingCascade Associated with Neuropathic Pain

Many animal models for neuropathic pain, such as chronic neuropathicpain, have been developed and characterized, see e.g., Bennett & Xie,Pain, vol. 33, 87-107 (1988); Seltzer et al., Pain, vol. 43, 205-18(1990); Kim & Chung, Pain, vol. 50, 355-63 (1992); Malmberg & Basbaum,Pain, vol. 76, 215-22 (1998); Sung et al., Neurosci Lett., vol. 246,117-9 (1998); Lee et al., Neuroreport, vol. 11, 657-61 (2000); Decosterd& Woolf, Pain, vol. 87, 149-58 (2000); Vadakkan et al., J Pain, vol. 6,747-56 (2005), each of which is hereby incorporated by reference in itsentirety. For a review of animal models used for neuropathic pain, seee.g., Eaton, J. Rehabilitation Research and Development, vol. 40 (4Supplement):41-54 (2003), the contents of which are hereby incorporatedby reference in their entirety.

Compounds that modulate neuropathic pain are identified using any of theart-recognized models for neuropathic pain. For example, the models forneuropathic pain generally involve injury to the sciatic nerve, althoughthe method used to induce injury varies. For example, the sciatic nerveis injured due to partial constriction, complete transection, freezingof the nerve, and metabolic, chemical, or immune insults to the nerve.Animals with these types of nerve injury have been shown to developabnormal pain sensations similar to those reported by neuropathic painpatients. In the studies described herein, the sciatic nerve of test andcontrol groups of subjects, such as mice, are injured. In the testgroup, subjects are administered a compound of the invention at avariety of times prior to, during and after injury to the sciatic nerve.The effects of the compound on the test group are compared to theeffects observed in the control group, e.g., through physicalobservation and examination of the subjects. For example, in mice, thesubject's hindpaw is used to test the response to non-noxious stimuli,such as tactile stimulation, or to test the subject's response tostimuli that would be noxious in the course of ordinary events, forexample, radiant heat delivered to the hindpaw. Evidence of allodynia, acondition in which ordinarily nonpainful stimuli evoke pain, or ahyperalgesia, the excessive sensitiveness or sensibility to pain, in thetest subjects indicates that test compound is not effectively modulatingneuropathic pain in the test subjects.

Example 17: Identification of Compounds that Modulate Kinase SignalingCascade Associated with Hepatitis B

Many animal models for hepatitis B have been developed andcharacterized. For a review of animal models of hepatitis B, see e.g.,Guha et al., Lab Animal, vol. 33(7):37-46 (2004), which is herebyincorporated by reference in its entirety. Suitable animal modelsinclude, for example, the chimpanzee, tree shrews (non-rodent smallanimals that are phylogenetically close to primates, see Walter et al.,Hepatology, vol. 24(1):1-5 (1996), which is hereby incorporated byreference in its entirety), and surrogate models such as the woodchuck,duck and ground squirrel. (See e.g., Tennant and Germ, ILAR Journal,vol. 42(2):89-102 (2001), which is hereby incorporated by reference inits entirety).

For example, primary hepatocytes are isolated from livers of the treeshrew species Tupaia belangeri and are infected with HBV. In vitroinfection results in viral DNA and RNA synthesis in hepatocytes andsecretion hepatitis B surface antigen (HBsAg) and hepatitis B e antigen(HBeAg) into culture medium. Tupaias can also be infected with HBV invivo, resulting in viral DNA replication and gene expression in Tupaialivers. Similar to acute, self-limited hepatitis B in humans HBsAg israpidly cleared from serum, followed by seroconversion to anti-HBe andanti-HBs.

Compounds that modulate hepatitis B are identified using any of theart-recognized models for hepatitis B. In the studies described herein,test and control groups of animals, e.g., chimpanzees or tree shrews,are infected with HBV. In the test group, subjects are administered acompound of the invention at a variety of times prior to, during andafter exposure to HBV. The effects of the compound on the test group arecompared to the effects observed in the control group, e.g., throughphysical observation and examination of the subjects and through bloodor serum analysis to determine at what point in time the infection iscleared from the subject. For example, assays are run to detect thepresence and/or amount of hepatitis B virus called surface antigens andfragments thereof. Alternatively or in addition, the subject's liver isanalyzed. Liver function tests analyze levels of certain proteins andenzymes, such as, for example, aspartate aminotransferase (AST, formerlyserum glutamic-oxaloacetic transaminase (SGOT)) and alanineaminotransferase (ALT, formerly serum glutamate-pyruvate transaminase(SGPT)).

Example 18: The Effect of Compounds on Tyrosine Kinase Inhibition

The following example illustrates that the compounds of the presentinvention could be used to treat autoimmune diseases. The compounds aretested using a method described previously (Goldberg, et al.; 2003, JMed. Chem., 46, 1337-1349). The kinase activity is measured using DELFIA(dissociation enhanced lanthanide fluoroimmunoassay), which utilizeseuropium chelate-labeled anti-phosphotyrosine antibodies to detectphosphate transfer to a random polymer, poly-Glu4-Tyr1 (PGTYR). Thekinase assay is performed in a neutravidin-coated 96-well white plate inkinase assay buffer (50 mM HEPES, pH 7.0, 25 mM MgCl2, 5 mM MnCl2, 50 mMKCl, 100 μM Na3VO4, 0.2% BSA, 0.01% CHAPS). Test samples (compounds ofthe instant invention) initially dissolved in DMSO at 1 mg/mL areprediluted for dose response (10 doses with starting final concentrationof 1 μg/mL, 1-3.5 serial dilutions) with the assay buffer. A 25 μLaliquot of this diluted sample and a 25 μL aliquot of diluted enzyme(lck) (0.8 nM final concentration) are sequentially added to each well.The reaction is started with a 50 μL/well of a mixture of substratescontaining 2 μM ATP (final ATP concentration is 1 μM) and 7.2 ng/μLPGTYR-biotin in kinase buffer. Background wells are incubated withbuffer and substrates only. Following 45 min of incubation at roomtemperature, the assay plate is washed three times with 300 μL/wellDELFIA wash buffer. A 100 μL/well aliquot of europium-labeledanti-phosphotyrosine (Eu³⁺-PT66, 1 nM, Wallac CR04-100) diluted inDELFIA assay buffer is added to each well and incubated for 30 min atroom temperature. Upon completion of the incubation, the plate is washedfour times with 300 μL/well of wash buffer and 100 μL/well of DELFIAwash buffer. Enhancement solution (Wallac) is added to each well. After15 min, timeresolved fluorescence is measured on the LJL's analyst(excitation at 360 nm, emission at 620 nm, EU 400 dichroic mirror) aftera delay time of 250 μs. The compound of the instant invention couldinhibit the kinase activity of lck, indicating that the compound may beused to treat autoimmune disease in a subject.

Example 19: IC₅₀ of KXO1 and Dasatinib in Dasatinib-resistant CellLines; Twelve (12) Concentrations of Inhibitor in Each Cell Line

Cancer cell lines reported in current literature to beDasatinib-resistant (i.e., COLO-320DM, H460, H226, and HCT-116) werecultured in the presence of the KXO1/KX2-391 Src inhibitor or Dasatinibcontrol in order to determine the effect of KXO1/KX2-391 on cell growthinhibition. Cell proliferation/growth inhibition was assessed using MTTcolorimetric assay. Additionally, the IC₅₀ of both KXO1/KX2-391 andDasatinib control was determined. Table 9 provides a list of the celllines used in this growth inhibition study.

TABLE 9 NAME ATCC No. TYPE H460 HTB-177 NSCLC H226 CRL-5826 NSCLCCOLO-320DM CCL-220 colorectal adenocarcinoma HCT116 CCL-247 colorectalcarcinoma

COLO-320DM, H460, H226, and HCT-116 human cancer cell lines wereroutinely cultured and maintained in basal medium containing 2% FBS at370 C, 5% CO₂. For the experiments, cells are seeded at 4.0×10^(3/190)μL and 8.0×10^(3/190) μL per well of 96-well plate in basal medium/1.5%FBS. Cells cultured are overnight (16 h) in 96-well plates at 37° C. inappropriate CO₂ conditions prior to KXO1/KX2-391 and Dasatinib addition.

For KXO1/KX2-391 and Dasatinib (BMS354825) dilutions, 20 mM stocksolution samples were diluted serially in basal medium/1.5% FBS using1:3 dilutions, yielding 20× concentrations in the 131 μM to 0.74 nMrange. 10 μL of 20× dilutions are then added to appropriate wells (n=3)containing 190 μL cancer cell lines, yielding 6561 nM to 0.037 nM rangeof final concentrations. The following controls were used: Vehiclecontrol of cells and no sample; Medium Control of cells, no sample, and0.03% DMSO (highest DMSO concentration present in samples; not reportedin results).

Treated cancer cells were incubated for 3 Days (78 hours) at 37° C.,appropriate CO₂ conditions. On Day 3, 10 μL MTT (5 mg/mL) was added toeach well. Cells were then incubated in the presence of MTT for 4 hoursat 37° C., appropriate CO₂ conditions. After this incubation period, 90μL 10% SDS(+HCl) was added to each well to lyse cells and solubilizeformazan. Cells were then incubated overnight at 37° C., appropriate CO₂conditions.

The OD₅₇₀ was measured using microplate reader. Growth inhibition curvesand EC₅₀/IC₅₀ was determined using GraphPad Prism 4 statisticalsoftware. Data was normalized to represent percent of maximum responseand is shown in FIGS. 24, 26, 28, and 30. FIGS. 25, 27, 29, and 31provides the data in pure OD₅₇₀ signal format.

Table 10 shows the IC₅₀s of KXO1/KX2-391 and Dasatinib in cancer celllines (8.0×10³ cells/well, 1.5% FBS) at 78 Hr (results from normalizedresponse data).

TABLE 10 Human Solid Tumor Cell Line KXO1 nM Dasatinib nM Dasatinib nMNAME IC₅₀ IC₈₀ IC₉₀ IC₅₀ IC₈₀ IC₉₀ Literature IC₅₀ H460 51 105 162 907,110 48,880  1,800*  H226 98 277 490 163 7,758 34,340 10,000*  COLO- 3080 144 1 2 14 10,000** 320DM HCT116 31 106 195 880 NA NA  5,000***Johnson et al., Clin Cancer Res 2005; 11(19): 6924-6932, Oct.1, 2005**Puputti et al., Mol Cancer Ther 2006; 5 (12): 927-934, December 2006

Example 20: Effect of KXO1/KX2-391 on Dasatinib and Imatinib ResistantLeukemia Cells

Ba/F3 cells (See e.g., Palacios et al., Nature 309: 126-131 (1984);Palacios et al., Cell 41: 727-734 (1985)) were cultured in 96-wellplates in complete media+IL-3. Cultures of Ba/F3 cells were alsotransfected to express wild-type (WT) Bcr-Abl, E255K mutation ofBcr-Abl, or T3151 mutation of Bcr-Abl and cultured in 96-well plates incomplete media without IL-3. The Ba/F3 cell line is rendered Gleevecresistant when the mutation in the Bcr/Abl tyrosine kinase E225K ispresent. The Ba/F3 cell line is rendered both Gleevec and Dasatinibresistant when the Bcr/Abl tyrosine kinase T3151 mutation is present.The cells of each group were then treated with no drug, 0.1-10,000 nMDasatinib, or 0.1-10,000 nM KXO1 in 10-fold dilutions for 96 hrs. MTTassays were performed (plate read at 570 nM). All assays are intriplicate.

The results of this study, summarized in FIGS. 32-33 and Table 11 below,shows that KXO1 inhibits the T3151 mutant of BCR-Abl at GI₅₀=35, whereasDasatinib does not inhibit at 10,000 nM. Further, Dasatinib does notinhibit IL-3-induced proliferation of Ba/F3 cells whereas KXO1 is apotent inhibitor (GI₅₀=3.5 nM).

TABLE 11 GI₅₀ values (nM) Cell line Dasatinib KXO1 Ba/F3 —   3.5 + WTBCR-Abl     1 85  + E225K     1 80  + T3151 >10,000 35 

Example 21: GI₅₀s/BrdU Assay in Five Cell Lines with KX2-391 andBMS354825

Evaluation of the GI₅₀s in five cell lines (SKOV-3, K562, HT-29, A549 &MDA-MB-231) with KX2-391 (KXO1 or Compound 134) and BMS354825 assayed atT=0 and T=72 using BrdU.

For these experiments, cells were seeded in two 96-well plates per cellline with the cell number indicated below in 200 μL growth mediacontaining 1.5% FBS. Cell lines being evaluated are: SKOV-2, K562,HT-29, A549, and MDA231. All seeded at 1000 cells per well except HT-29(2000 cells) and MDA MB 231 (5000 cells). The plates were incubated for24 hours after seeding at 37° C.+5% CO₂. Except MDA231, this line isgrown at 37° C. and 0% CO₂.

After 24 hours post-seeding, KX2-391 and BMS354825 were added at 128 nM,64 nM, 32 nM, 16 nM, 8 nM, 4 nM, 2 nM, and 1 nM to 1 plate of each cellline (n=3). The KX2-391 and BMS354825 treated sets of cell line plateswere incubated for 72 hours at 37° C.+5% CO₂. Except MDA231, this lineis grown at 37° C. and 0% CO₂. Brdu assay was performed at T=0 and T=72.

Growth Inhibition. The BrdU data was used to determine the % growthinhibition for each sample concentration using the formula:

GI = [(T₁ − T₀)/(Con − T₀)] × 100

where T₀=Fluorescence of cells at time 0; T₁=Fluorescence of treatedcells at x hours; Con=Fluorescence of control cells at x hours. T₁values≤T₀ values were designated as T, cytotoxicity. The GI₅₀ wasestimated using XLFit excluding T₁ values≤T₀ (cytotoxicity). The resultsof this study, summarized in FIGS. 36-40 and Table 12 below.

TABLE 12 GI₅₀ Data Summary KX2-391 BMS-354825 HT-29 1.54E−08 M 2.05E−08M SKOV-3 9.75E−09 M 3.24E−09 M A549 9.39E−09 M 1.25E−08 M K562 1.09E−08M <1.0E−9 M  MDA-MB-321 1.98E−08 M 6.02E−09 M

Example 22: Combination GI₅₀ of Gemzar and (KXO1/KX2-391) in the L3.6plCell Line Using the BrdU Assay

This study involved the evaluation of the GI₅₀ of Gemzar±KX2-391 in theL3.6pl cell line assayed at T=0 and T=72 using the BrdU Assay (Roche:Catalog Number, 11647229001). L3.6pl cells, a human pancreatic cancercell line, were seeded in three 96-well plates with 2000 cells/well forL3.6pl in 190 μL growth media containing 1.5% FBS. L3.6pl cells arepreviously described in Trevino et al. Am J Pathol. 2006 March;168(3):962-72, hereby incorporated herein by reference in its entirety.The cells were incubated for 18-24 hours after seeding at 37° C.+5% CO₂.After 24 hours, Gemzar+KX2-391, Gemzar, and KX2-391 was added to theL3.6pl cells (n=3). Gemzar was evaluated at concentrations of 8 nM, 4nM, 2 nM, 1 nM, 0.5 nM, 0.25 nM, 0.125 nM, 0.063 nM. KX2-391 wasevaluated at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM,3.125 nM, 1.56 nM, and 0.78 nM. Each sample treated plate was incubatedfor 72 hours at 37° C.+5% CO₂. The BrdU assay was performed at T=0 andagain after 72 hours of incubation, T=72. The results of the study areprovided in FIGS. 34 and 35. Table 13 provides a summary of thecalculated GI₅₀ for Gemzar±KX2-391.

TABLE 13 GI₅₀ Summary Table KX2-391 nM Gemzar nM Single 53.03 1.76Combined  1.15 0.09

Example 23: Orthotopic Prostate Model for Measuring in Vivo Metastases

Nu/Nu mice (8-12 weeks of age) were injected with PC3-MM2 prostatecancer cells into the prostate as described previously in Pettaway etal., Clin Cancer Res 1996, 2:1627-1636, hereby incorporated herein byreference in its entirety. Fourteen days after orthotopic injection ofPC3-MM2 cells, the mice were randomized into four groups: Dasatinib (15mg/kg/day) treatment; KXO1 (5 mg/kg/day) treatment; KX2-391 (10mg/kg/day) treatment; and control (vehicle). Dasatinib, KXO1, andvehicle was administered by oral gavage. All mice were sacrificed bycervical dislocation on about day 42. Tumor volume (measured bycaliper), weight, and incidence of regional (celiac or para-aortal)lymph node metastases were recorded. Results of the experiment arereported in Table 14 and shown in FIGS. 41 and 47.

TABLE 14 Tumor Tumor weight (g) LN incidence Median (IQR) metastasisControl 5/6 2.27 (1.94 ~ 2.61) 5/5 KX 01 (5.0mg/kg/day) 5/6 1.16 (0.94 ~1.28) 4/5 KX 01 (10.0mg/kg/day) 5/6 0.35 (0.24 ~ 0.56) 2/5 Dasatinib(15mg/kg/day) 5/6 0.43 (0.30 ~ 1.34) 2/5

Example 24: HBV Primary Assay

The HBV primary assay developed was conducted similarly to thatdescribed by Korba et al., (Antiviral Res. 15: 217-228 (1991) andAntiviral Res. 19: 55-70 (1992)) with the exception that viral DNAdetection and quantification have been improved and simplified (Korba etal., Antiviral Res. 19: 55-70 (1992)).

Compound 38 (KX2-377), Compound 136 (KX2-393), and Compound 137(KX2-394) were evaluated for potential anti-HBV activity using a singlehigh-test concentration of compounds in the standardized HepG2-2.2.15antiviral assay. The HepG2-2.2.15 is a stable cell line producing highlevels of the wild-type ayw1 strain of HBV. Briefly, HepG2-2.2.15 cellswere plated in 96-well plates. Only the interior wells were utilized toreduce “edge effects” observed during cell culture; the exterior wellsare filled with complete medium to help minimize sample evaporation. Onthe following day, the confluent monolayer of HepG2-2.2.15 cells waswashed and the medium is replaced with complete medium containing testconcentrations of a test article in triplicate. 3TC was used as thepositive control, while media alone was added to cells as the untreatedcontrol. Three days later the culture medium was replaced with freshmedium containing the appropriately diluted drug. Six days following theinitial administration of the test compound, the cell culturesupernatant was collected, treated with pronase and DNAse and then usedin a real-time quantitative TaqMan PCR assay for direct measurement ofHBV DNA copies using an ABI Prism 7900 sequence detection system(Applied Biosystems, Foster City, Calif.).

The antiviral activity of each test compound was calculated by comparingits HBV DNA copies against that of the untreated control cells (100%) toderive percent inhibition level. After removing the supernatant, theremaining cells were subject to CellTiter 96 Aqueous One (Promega,Madison, Wis.) solution cell proliferation assay (MTS-based) to measurecell viability. Cytotoxicity of the compound was determined by comparingits cell viability with that of the untreated cell control to derivepercentage of the cell control. Results of this study are provided inTable 15 below and in FIG. 42.

TABLE 15 Antiviral Activity Cytotoxicity Percent Percent Test inhibitionof of cell Compound Concentration cell control control Interpretation3TC  1 μM 92.0% 103.3%  Active KXO1:2HCl (KX2-391) 10 μM 48.2% 51.3%Cytotoxic KX0-D (KX2-392) 10 μM 83.3% 81.2% Active KX0-E (KX2-394) 10 μM62.3% 32.2% Cytotoxic KX0-C 10 μM  69.62% 94.5% Active (KX2-377)

Example 25: Inhibition of Src Kinase Activity in Whole Cells

The compounds of the invention inhibit Src kinase activity in wholecells as shown in FIGS. 44A, 44B, 44C, and 44D. FIG. 44A is a graphdepicting the effect of KXO1 on Src autophosphorylation in c-Src/NIH-3T3cells; FIG. 44B is a graph indicating the effect of KXO1 on Srcautophosphorylation in HT-29 cells; FIG. 44C is a graph depicting theeffect of KXO1 on Src transphosphorylation in c-Src/NIH-3T3 cells; andFIG. 44D is a graph indicating the effect of KXO1 on Srcautophosphorylation in HT-29 cells. KXO1 is a potent inhibitor of Srckinase activity in whole cells. As shown in FIGS. 44A-44D, KXO1 is apotent inhibitor of Src kinase activity in whole cells. In particular,KXO1 was a potent inhibitor of Src autophosphorylation (FIGS. 44A and44B) and Src transphosphorylation (FIGS. 44C and 44D) in various celllines. Similar whole cell inhibition results were obtained foradditional transphosphorylation substrates, i.e., FAK Y925 & paxillinY31. Phosphorylations of PDGF Y572/574, EGF Y845, JAK1 Y1022/1023 & JAK2Y1007/1008, Lck Y405 & ZAP70 Y319 were not inhibited in whole cells. LynY416 and Bcr/Abl &245 were inhibited less potently.

Example 26: Selectivity for Protein Tyrosine Kinases in Whole Cells

The compounds of the invention are selective for protein tyrosinekinases (PTKs). FIG. 45 is an illustration depicting the selectivity ofKXO1 for protein tyrosine kinases (PTKs) in whole cells as compared toDasatinib, an ATP-competitive Src inhibitor currently in clinicaltrials. SYF cells are mouse fibroblasts that lack the Src kinase familymembers Src, Yes and Fyn. KXO1 demonstrated very high PTK selectivity inwhole cells as compared to Dasatinib.

Example 27: Oral Potency

The compounds of the invention demonstrate high oral potency. Forexample, FIG. 46 shows the oral potency of KXO1 in comparison toDasatinib. Oral potency was determined using staged HT29 (human coloncancer) mouse Xenografts over a period of 28 days of treatment KXO1 wastested at 2.0 and 4 mg/kg bid. Dasatinib was tested at 25 mg/kg bid. Atday 5, Dasatinib dose was lowered to 15 mg/kg bid due to weight loss.

Example 28: HCV Primary Assay

The compounds of the invention could be used to treat HCV. The compoundsare tested using the method of Pietschmann, T., et al. J. Virol.76:4008-4021. The ET call line is a human hepatoma cell line, Huh-7,harboring an HCV RNA replicon (genotype 1b) with a stable luciferase(Luc) reporter and three cell culture-adaptive mutations. The cells aregrown in Dulbecco's modified essential media (DMEM), 10% fetal bovineserum (FBS), 1% penicillin-streptomycin (pen-strep), 1% glutamine, 5mg/ml G418 in a 5% CO₂ incubator at 37° C. All cell culture reagents arefrom e.g., Mediatech (Herndon, Va.).

Example 29: Bioavailability

The compounds of the invention demonstrate good bioavailability. Forexample, the bioavailability and pharmacokinetic parameters of compound139

are described below. Plasma concentrations were measured in mice afteroral and IV administration. Both oral and IV doses were formulated insaline. Male CD-1 mice were dosed after an overnight fast and fed 4hours post-dose.

IV Administration

A single IV dose of 139 was administered to male CD-1 mice intravenouslyat 5 mg/kg (dose volume of 3 mL/kg). The plasma concentration of 139 wasmeasured at 0 min, 5 min, 15 min, 30 min, 1 h, 3 h, 6 h, and 12 h timepoints. A liquid-liquid extraction procedure was used to isolate theanalyte and internal standard from mice plasma. After the extract wasdried and reconstituted, the sample was directly injected into a liquidchromatography/mass spectrometry (LC/MS) system. HPLC conditions were asfollows:

-   -   HPLC System: Shimadzu SCL-10 System    -   Analytical Column: Aquasil C18 5 μm 50×2.1 mm column.    -   Column Temperature: Ambient temperature    -   Autosampler Temperature: Ambient temperature    -   Mobile Phase A) 10 mM Ammonium formate in water (pH4).        -   B) Acetonitrile.    -   Flow Rate: 0.6 mL/min    -   Injection Volume: 4 μL    -   Gradient:

Time (Minute) 0 2.5 3.5 4.2 4.3 4.5 4.7 5 5.3 7.5 % B 18 18 75 75 18 1895 95 18 stopMass spectrometry conditions were as follows:

Instrument: ABI Sciex API 4000 Mode: ESI+

Experiment: MRM (multiple reaction monitoring)Transitions: 139: m/z 480.1→127.1 (Rt=3.8 minute)

Table 16 shows the plasma concentration of 139 over a 12 hour period andTable 17 shows the resulting pharmacokinetic parameters. Compound 139showed a half-life of 1.6 hours. The time points used for the estimationof t/2 were 3, 6, and 12 hours.

TABLE 16 Compound 139 Plasma Concentrations (ng/mL) in Male CD-1 MiceAfter a Single IV Dose of 5 mg/kg Time Group A Group B Group C Mean SD %CV 0 BLQ BLQ BLQ 0.00 0.00 NA 5 min 1195 1287 1260 1247 47.29 3.79 15min 273 306 407 329 69.82 21.22 30 min 500 367 279 382 111.26 29.13 1 hr186 210 137 178 37.21 20.90 3 hr 124 332 164 207 110.37 53.32 6 hr 65.320.0 44.2 43.2 22.67 52.48 12 hr 11.0 BLQ BLQ 3.67 6.35 173.02 NA: NotApplicable. BLQ: Below Limit of Quantitation (10 ng/mL) BLQ = 0 whencalculating mean, SD and % CV

TABLE 17 Pharmacokinetic Parameters of Compound 139 in Male CD-1 MiceAfter a Single IV Dose of 5 mg/kg % AUC Sample C₀ t_(1/2) AUC_(last)AUC_(0−∞) AUC_(0−∞)/Dose Extrap Vss CL MRT ID (ng/mL) (hr) (ng · hr/mL)(ng · hr/mL) (hr · kg · ng/mL/mg) (%) (L/kg) (mL/min/kg) (hr) Plasma2420 1.6 1412 1420 284.1 0.6 81.52 58.67 2.3

Oral Administration

A single IV dose of 139 was administered to male CD-1 mice oral at 25mg/kg (dose volume of 10 mL/kg). The plasma concentration of 139 wasmeasured at 0 min, 30 min, 1 h, 3 h, 6 h, 12 h, and 24 h time points. Asdiscussed above for the i.v. administration, the analyte was extractedand analyzed by HPLC/mass spec.

Table 18 shows the plasma concentration of 139 over a 24 hour period andTable 19 shows the resulting pharmacokinetic parameters. Compound 139showed a half-life of 6.2 hours. The time points used for the estimationof t_(1/2) were 6, 12, and 24 hours.

TABLE 18 Compound 139 Plasma Concentrations (ng/mL) in Male CD-1 MiceAfter a Single PO Dose of 25 mg/kg Time Group A Group B Group C Mean SD% CV 0 BLQ BLQ BLQ 0.00 0.00 NA 30 min 310 89.6 554 318 232.30 73.05 1hr 157 211 58.9 142 77.11 54.30 3 hr 641 220 130 330 272.78 82.61 6 hr80.6 184 92.0 119 56.69 47.64 12 hr 31.7 106 32.0 56.6 42.81 75.64 24 hr31.2 15.6 BLQ 15.6 15.60 100.00 NA: Not Applicable. BLQ: Below Limit ofQuantitation (10 ng/mL) BLQ = 0 when calculating mean, SD and % CV

TABLE 19 Pharmacokinetic Parameters of Compound 139 in Male CD-1 MiceAfter a Single Oral Dose of 25 mg/kg % AUC Sample t_(1/2) C_(max)T_(max) AUC_(last) AUC_(0−∞) AUC_(0−∞)/Dose Extrap MRT ID (hr) (ng/mL)(hr) (ng · hr/mL) (ng · hr/mL (hr · kg · ng/mL/mg) (%) (hr) Plasma 6.2330 3.0 2300 2439 97.6 5.7 7.7

Estimated bioavailability for Compound 139 is

(Mean AUC_(0-∞) oral/oral dose)/(Mean AUC_(0-∞) IV/IV dose)×100%=34.4%

Example 30: Plasma and Brain Exposure

The compounds of the invention demonstrate good plasma/brain exposure.For example, the plasma and brain exposure of KXO1 (also referred to asKX2-391 and compound 134) and compound 76 (also referred to as KX2-361)are described below. Plasma concentrations were measured in mice afteroral administration. All doses were formulated in purified water. Fourgroups of male CD-1 mice were dosed after an overnight fast and fed 4hours post-dose. Dosing was as follows:

Group Dose Dose Vol. Number Route Compound (mg/kg)* (mL/kg) 1 PO KX2-391Mesylate 10 10 2 PO KX2-391 Mesylate 50 10 3 PO KX2-361 2HCl 10 10 4 POKX2-361 2HCl 50 10 *Note: Doses administered were mg free base/kg

Protein was precipitated with 0.25 mL acetonitrile for plasma, 0.25 mLfor brain. After centrifugation, supernatant was directly injected intoan LC/MS system. The limit of quantitation was 1 ng/mL using a 50 μLaliquot for plasma and a 50 μL aliquot for brain. The standard curve was1 to 1,000 ng/mL for both plasma and brain.

HPLC conditions were as follows:

-   -   HPLC System: Shimadzu SCL-10 System    -   Analytical Column: Aquasil C18 5 μm 100×2 mm column.    -   Column Temperature: Ambient temperature    -   Autosampler Temperature: Ambient temperature    -   Mobile Phase A) 10 mM Ammonium formate in water (pH 4).        -   B) Acetonitrile.    -   Flow Rate: 0.6 mL/min    -   Injection Volume: 2 μL    -   Gradient:

Time (Minute) 0.0 1.6 2.6 3.8 3.9 4.1 4.4 4.6 4.65 7.0 % B 20 20 65 6520 20 95 95 20 StopMass Spectrometry Conditions were as follows:

Instrument: ABI Sciex API 4000 Mode: ESI+

Experiment: MRM (multiple reaction monitoring)Transitions: KX2-391: m/z 432.4→114.2 (Rt=3.11 minute)

-   -   KX2-361: m/z 406.1→253.2 (Rt=3.33 minute)

Tables 20-24 below show plasma and brain concentrations following theadministration of a single oral dose of KX2-391 at 10 mg/kg and 50mg/kg.

TABLE 20 KX2-391 Plasma Concentrations (ng/mL) in Male CD-1 Mice After aSingle PO Dose of 10 mg/kg (Group 1) Time (hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 778.51 1096.62 737.37 870.83196.62 22.58 1 516.97 328.28 243.96 363.07 139.79 38.50 2 328.47 271.89261.57 287.31 36.02 12.54 5 133.38 147.74 160.62 147.25 13.63 9.26 NA:Not Applicable. BLQ: Below Limit of Quantitation (1 ng/mL) BLQ = 0 whencalculating mean, SD and % CV

TABLE 21 KX2-391 Brain Concentrations (ng/g) in Male CD-1 Mice After aSingle PO Dose of 10 mg/kg (Group 1) Time (hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 398.43 509.00 286.70 398.04111.15 27.92 1 150.70 266.66 92.06 169.81 88.85 52.32 2 125.69 84.0485.88 98.54 23.53 23.88 5 67.68 75.21 71.22 71.37 3.77 5.28

TABLE 22 KX2-391 Plasma Concentrations (ng/mL) in Male CD-1 Mice After aSingle PO Dose of 50 mg/kg (Group 2) Time (hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 8511.88 8334.53 12315.319720.57 2248.85 23.13 1 2374.12 2442.20 1365.56 2060.62 602.91 29.26 21148.57 1546.09 1850.18 1514.95 351.84 23.22 5 424.48 1139.11 1201.91921.83 431.86 46.85

TABLE 23 KX2-391 Brain Concentrations (ng/g) in Male CD-1 Mice After aSingle PO Dose of 50 mg/kg (Group 2) Time (hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 2795.27 3190.42 5089.323691.67 1226.42 33.22 1 945.72 936.29 482.22 788.08 264.92 33.62 2613.18 530.41 684.97 609.52 77.34 12.69 5 200.01 387.73 522.17 369.97161.81 43.74

The brain and plasma pharmacokinetic parameters of KX2-391 in mice aftera single dose of 10 mg/kg (Group 1) are as follows:

Sample T_(max) C_(max) AUClast ID (hr) (ng/mL)) (ng · hr/mL) Brain 0.50398 631 Plasma 0.50 871 1503 Note: Brain Cmax and AUClast are ng/g andng · hr/g, respectively.

The AUClast Brain/AUClast Plasma Ratio is 0.42.

The brain and plasma pharmacokinetic parameters of KX2-391 in mice aftera single dose of 50 mg/kg (Group 2) are as follows:

Sample T_(max) C_(max) AUClast ID (hr) (ng/mL) (ng · hr/mL) Brain 0.503692 4211 Plasma 0.50 9721 10818 Note: Brain Cmax and AUClast are ng/gand ng · hr/g, respectively.

The AUClast Brain/AUClast Plasma Ratio is 0.39.

Tables 25-28 below show plasma and brain concentrations following theadministration of a single oral dose of KX2-361 at 10 mg/kg and 50mg/kg.

TABLE 25 KX2-361 Plasma Concentrations (ng/mL) in Male CD-1 Mice After aSingle PO Dose of 10 mg/kg (Group 3) Time (hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 65.93 217.56 159.53 147.6776.51 51.81 1 284.09 596.31 398.35 426.25 157.97 37.06 2 153.07 118.96342.45 204.83 120.40 58.78 5 2.63 64.15 53.18 39.99 32.81 82.05 NA: NotApplicable. BLQ: Below Limit of Quantitation (1 ng/mL) BLQ = 0 whencalculating mean, SD and % CV

TABLE 26 KX2-361 Brain Concentrations (ng/g) in Male CD-1 Mice After aSingle PO Dose of 10 mg/kg (Group 3) Time(hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 43.98 138.55 116.84 99.7949.54 49.64 1 227.97 334.71 273.32 278.67 53.57 19.22 2 109.78 78.76336.07 174.87 140.46 80.32 5 BLQ 47.45 47.85 31.77 27.51 86.59

TABLE 27 KX2-361 Plasma Concentrations (ng/mL) in Male CD-1 Mice After aSingle PO Dose of 50 mg/kg (Group 4) Time(hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 1488.55 2316.91 1587.181797.54 452.48 25.17 1 1805.19 969.48 3635.91 2136.86 1363.81 63.82 2475.13 1710.13 1709.02 1298.09 712.70 54.90 5 138.53 252.81 298.99230.11 82.60 35.90

TABLE 28 KX2-391 Brain Concentrations (ng/g) in Male CD-1 Mice After aSingle PO Dose of 50 mg/kg (Group 4) Time(hr) Group A Group B Group CMean SD % CV 0 BLQ BLQ BLQ 0.00 0.00 NA 0.5 960.08 2124.87 1051.161378.70 647.80 46.99 1 850.96 1888.05 548.55 1095.85 702.53 64.11 2413.89 985.90 874.17 757.99 303.19 40.00 5 121.56 260.41 238.66 206.8874.68 36.10

The brain and plasma pharmacokinetic parameters of KX2-361 in mice aftera single dose of 10 mg/kg (Group 3) are as follows:

Sample T_(max) C_(max) AUClast ID (hr) (ng/mL) (ng · hr/mL) Brain 1.00279 656 Plasma 1.00 426 863 Note: Brain Cmax and AUClast are ng/g and ng· hr/g, respectively.

The AUClast Brain/AUClast Plasma Ratio is 0.76.

The brain and plasma pharmacokinetic parameters of KX2-361 in mice aftera single dose of 50 mg/kg (Group 4) are as follows:

Sample T_(max) C_(max) AUClast ID (hr) (ng/mL) (ng · hr/mL) Brain 0.501379 3338 Plasma 1.00 2137 5443 Note: Brain Cmax and AUClast are ng/gand ng · hr/g, respectively.

The AUClast Brain/AUClast Plasma Ratio is 0.61.

Example 31: Glioma Survival Studies

A brain tumor mouse xenograft study was conducted comparing KX2-361 andKX2-391 (KXO1) to Temodar®. The studies were conducted in C57BL/6 mice.GL261 glioma cells (1×10⁵ in 5 μl DMEM) were implanted intracranialcoordinates: bregma, lateral 2.0 mm, anterior 1.2 mm, 3.0 mm depth dura.Treatment was initiated 3 days post implantation. The groups were asfollows (all doses in 100 μl H₂O):

Vehicle (H₂O) KX2-391 2.5 mg/kg bid oral KX2-391 5 mg/kg bid oralKX2-361 15 mg/kg bid oral KX2-361 30 mg/kg bid oral Temodar ® 5 mg/kgonce weekly oral

Table 29 below shows a summary of the results. The median survival rangeand the log-rank (Mantel-Cox) statistical test results comparing thesurvival distributions of the samples. KX2-361 exhibited the bestresults when administered orally at 15 mg/kg/dose bid 7 hours apart.

TABLE 29 Temodar ® KX2-391 KX2-391 KX2-361 KX2-361 5 mg/kg 2.5 mg/kg 5mg/kg 15 mg/kg 30 mg/kg weekly x1 Vehicle bid oral bid oral bid oral bidoral oral Median 22 25 23 30.5 29 29 survival 21-25 22-36 22-29 25-3423-32 26-29 Range: Log-Rank (Mantel-Cox) Test vs. P = 0.1062 P = 0.1762P = 0.0106 P = 0.0425 P = 0.0017 Vehicle vs. P = 0.0017 P = 0.3649 P =0.1366 P = 0.2396 P = 0.5237 Temodar vs. KX2- P = 0.7559 P = 0.6530 P =0.8901 391 2.5 mg/kg vs. KX2- P = 0.0605 P = 0.1166 P = 0.1366 391 5mg/kg

FIGS. 49A-E show the weight gain in each of the C57BL/6 mice in thedifferent treatment groups. The average weight at endpoint for each ofthe treatment groups is shown below in Table 30. FIG. 50 is a graphshowing the average weights over a 40-day period for each of thetreatment groups.

TABLE 30 Average weight at endpoint Vehicle 19.2 g KX2-361  15 mg/kg16.9 g KX2-361  30 mg/kg 15.0 g KX2-391 2.5 mg/kg 16.0 g KX2-391   5mg/kg 14.3 g Temodar ®   5 mg/kg 13.3 g

Example 32. Synergistic Cell Growth Inhibition Using a Combination

The combination KOX1 and tamoxifen was tested in vitro to determine theability of the combination to inhibit cell growth in MCF-7 breast cancercells. A range of concentrations was tested by MTT Assay as shown below.

KXO1 Tamoxifen (nM) (nM) Fa Cl 10 50 0.325758 0.819 10 100 0.47365 0.91525 50 0.3521 0.900 25 100 0.4937 0.906 50 50 0.3715 1.048 50 100 0.53260.852 75 100 0.6913 0.505 75 50 0.4196 0.948

The MTT cell growth data was analyzed by the CalcuSyn software(Biosoft). This program uses the median-effect principle (77) todelineate the interaction between two drugs. For each dose combination,the program generates a combination index (CI). A combination index (CI)of <1, 1 or >1 denotes synergism, additivity or antagonism respectively.FIG. 51 shows synergistic growth inhibitory effects with 100 nMtamoxifen+75 nM KXO1. The CI value for this combination was calculatedto be 0.505.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims. It will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the invention encompassed bythe appended claims.

1. Use of a compound according to Formula IB:

or a salt, solvate, hydrate, or prodrug thereof, wherein: T is a bond;X_(y) is CY, N, or N—O; X_(z) is CZ; Y is selected from hydrogen,hydroxyl, halogen, lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, C₁, C₂, C₃,C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl,and O-benzyl; X_(a) is CR_(a) or N, or N—O; X_(b) is CR_(b), N, or N—O;X_(c) is CR_(c) or N, or N—O; X_(d) is CR_(d) or N, or N—O; X_(e) isCR_(e), N, or N—O; R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆are, independently, hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅,or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄,C₅, or C₆) alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH,COOH, COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂-lower (C₁,C₂, C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower alkyl is linear orbranched alkyl; K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂,NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy,or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are C₁, C₂, C₃, C₄, C₅, or C₆ alkyl or R₁₉ and R₂₀taken together with the attached nitrogen atom form a five memberedring; V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—; R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently,H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; Z is(CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, R₁, R₂, andR₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; and n and mare independently 0, 1, or 2, in the manufacture of a medicament fortreating or preventing cancer, wherein the cancer is selected fromrenal, liver, or brain cancer. 2.-19. (canceled)
 20. A compositioncomprising tamoxifen and a compound of Formula I:

or a salt, solvate, hydrate, or prodrug thereof, wherein: T is a bond,CR₁₂R₁₃, C(O), O, S, S(O), S(O)₂, NR₁₄, C(R₁₅R₁₆)C(R₁₇R₁₈), CH₂O, orOCH₂; X_(y) is CZ, CY, N, or N—O; X_(z) is CZ, CY, N, or N—O; at leastone of X_(y) and X_(z) is CZ; Y is selected from hydrogen, hydroxyl,halogen, lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-aryl, and O-benzyl;X_(a) is CR_(a) or N, or N—O; X_(b) is CR_(b), N, or N—O; X_(c) isCR_(c) or N, or N—O; X_(d) is CR_(d) or N, or N—O; X_(e) is CR_(e), N,or N—O; R_(a), R_(b), R_(c), R_(d), R_(e), R₄, R₅, and R₆ are,independently, hydrogen, hydroxyl, halogen, P, C₁, C₂, C₃, C₄, C₅, or C₆alkyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkoxy, O-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-aryl, O-benzyl, C₁, C₂, C₃, C₄, C₅, or C₆ alkyl-OH, COOH,COO-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl, SO₂H, SO₂-lower (C₁, C₂,C₃, C₄, C₅, or C₆) alkyl,

where W is H, or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl, C₁, C₂, C₃, C₄, C₅, orC₆ alkyl-aryl; P is SO₃H, OSO₃H, OPO₃H₂, OPO₃H₂, NH₂, NHR₁₉, NHR₂₀R₂₁,

tetrazole, O-lower (C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-K, O—C(O)-lower(C₁, C₂, C₃, C₄, C₅, or C₆) alkyl-L, NH-lower (C₁, C₂, C₃, C₄, C₅, orC₆) alkyl-M, or O-aryl-Q, further wherein lower alkyl is linear orbranched alkyl; K is C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂, NH₂,NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆ alkoxy,or

L is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

M is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

Q is aryl, heteroaryl, OH, C(O)NH₂, COOH, SO₃H, OSO₃H, PO₃H₂, OPO₃H₂,NH₂, NHR₁₉, NR₁₉R₂₀, SO₂R₂₁, glycoside, lower C₁, C₂, C₃, C₄, C₅, C₆alkoxy, or

R₁₉, R₂₀ and R₂₁ are C₁, C₂, C₃, C₄, C₅, or C₆ alkyl or R₁₉ and R₂₀taken together with the attached nitrogen atom form a five memberedring; V is a bond, —CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —O—CH₂—, —OCH₂CH₂— or—OCH₂CH₂CH₂—; R₁₂, R₁₃, R₁₄, R₁₅, R₁₆, R₁₇, and R₁₈, are, independently,H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; Z is(CHR₁)_(n)—C(O)—NR₂(CHR₃)_(m)—Ar, where Ar is a substituted orunsubstituted aryl or nitrogen-containing heteroaryl group, R₁, R₂, andR₃ are independently H or C₁, C₂, C₃, C₄, C₅, or C₆ alkyl; and n and mare independently 0, 1, or 2.